Murine interferon-α

ABSTRACT

Interferons represent an important class of biopharmaceutical products, which have a proven track record in the treatment of a variety of medical conditions, including the treatment of certain autoimmune diseases, the treatment of particular cancers, and the enhancement of the immune response against infectious agents. The present invention provides a new form of murine interferon-α, which has applications in diagnosis and therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional application Nos.60/125,045 (filed Mar. 18, 1999), and 60/155,739 (filed Sep. 23, 1999),the contents of which are incorporated by reference.

TECHNICAL FIELD

The present invention relates generally to a new cytokine havingdiagnostic and therapeutic uses. In particular, the present inventionrelates to a novel murine interferon-α, and to nucleic acid moleculesencoding interferon-α.

BACKGROUND OF THE INVENTION

Cellular differentiation of multicellular organisms is controlled byhormones and polypeptide growth factors. These difflusable moleculesallow cells to communicate with each other and act in concert to formtissues and organs, and to repair and regenerate damaged tissue.Examples of hormones and growth factors include the steroid hormones,parathyroid hormone, follicle stimulating hormone, the interferons, theinterleukins, platelet derived growth factor, epidermal growth factor,and granulocyte-macrophage colony stimulating factor, among others.

Hormones and growth factors influence cellular metabolism by binding toreceptor proteins. Certain receptors are integral membrane proteins thatbind with the hormone or growth factor outside the cell, and that arelinked to signaling pathways within the cell, such as second messengersystems. Other classes of receptors are soluble intracellular molecules.

Of particular interest, from a therapeutic standpoint, are theinterferons (reviews on interferons are provided by De Maeyer and DeMaeyer-Guignard, “Interferons,” in The Cytokine Handbook 3^(rd) Edition,Thompson (ed.), pages 491-516 (Academic Press Ltd. 1998), and by Walsh,Biopharmaceuticals: Biochemistry and Biotechnology, pages 158-188 (JohnWiley & Sons 1998)). Interferons exhibit a variety of biologicalactivities, and are useful for the treatment of certain autoimmunediseases, particular cancers, and the enhancement of the immune responseagainst infectious agents, including viruses, bacteria, fungi, andprotozoa. To date, six forms of interferon have been identified, whichhave been classified into two major groups. The so-called “type I”interferons include interferon-α, interferon-β, interferon-ω,interferon-δ, and interferon-τ. Currently, interferon-γ and one subclassof interferon-α are the only type II interferons.

Type I interferons, which are thought to be derived from the sameancestral gene, have retained sufficient similar structure to act by thesame cell surface receptor. The α-chain of the human interferon-α/βreceptor comprises an extracellular N-terminal domain, which has thecharacteristics of a class II cytokine receptor. Interferon-γ does notshare significant homology with the type I interferons or with the typeII interferon-α subtype, but shares a number of biological activitieswith the type I interferons.

In humans, at least 16 non-allelic genes code for different subtypes ofinterferon-α, while interferons β and ω are encoded by single genes.Type I interferon genes are clustered in the short arm of chromosome 9.Unlike typical structural human genes, interferon-α, interferon-β, andinterferon-ω lack introns. A single gene for human interferon-γ islocalized on chromosome 12 and contains three introns. To date,interferon-τ has been described only in cattle and sheep, whileinterferon-δ has been described only in pigs.

At least 12 non-allelic murine interferon-α genes have been identifiedso far (for a review, see De Maeyer and De Maeyer-Guignard,“Interferons,” in The Cytokine Handbook, 3^(rd) Edition, Thompson (ed.),pages 491-516 (Academic Press Ltd. 1998)). In general, the structure ofmurine interferon-α genes is similar to that of corresponding humangenes. The mouse also appears to have a single interferon-β gene. Murineinterferon-α and -β genes are clustered on chromosome 4, although theinterferon-β gene is distal from the interferon-α cluster.

Clinicians are taking advantage of the multiple activities ofinterferons by using the proteins to treat a wide range of conditions.For example, one form of interferon-α has been approved for use in morethan 50 countries for the treatment of medical conditions such as hairycell leukemia, renal cell carcinoma, basal cell carcinoma, malignantmelanoma, AIDS-related Kaposi's sarcoma, multiple myeloma, chronicmyelogenous leukemia, non-Hodgkin's lymphoma, laryngeal papillomatosis,mycosis fungoides, condyloma acuminata, chronic hepatitis B, hepatitisC, chronic hepatitis D, and chronic non-A, non-B/C hepatitis. The U.S.Food and Drug Administration has approved the use of interferon-β totreat multiple sclerosis, a chronic disease of the nervous system.Interferon-γ is used to treat chronic granulomatous diseases, in whichthe interferon enhances the patient's immune response to destroyinfectious bacterial, fungal, and protozoal pathogens. Clinical studiesalso indicate that interferon-γ may be useful in the treatment of AIDS,leishmaniasis, and lepromatous leprosy.

Although new uses of known interferons may be discovered, a need existsfor the provision of new interferons for biopharmaceuticals.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a novel murine interferon-α, designated“Zcyto13.” The present invention also provides Zcyto13 polypeptides andZcyto13 fusion proteins, as well as nucleic acid molecules encoding suchpolypeptides and proteins, and methods for using these nucleic acidmolecules and amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

A nucleic acid molecule containing a sequence that encodes murineinterferon-α, initially designated as “Zcyto13,” has the followingnucleotide sequence:

ATACTAAGCA CCAGGGTTGA GAATGACTCC AAAGTTTTTA TGGCTGGTGG [SEQ ID NO:1].CCCTTGTGGC TCTATACATT CCGCCCATCC AATCTCTGAA CTGTGTTTAC CTGGATGATAGCATCTTGGA AAATGTGAAA CTTCTGGGCA GTACCATGAC CGGCTTTCCC TTAAGATGTCTAAAAGATAT CACAGATTTT AAGTTTCCTA AAGAGATTTT GCCATACATC CAGCATATGAAAAGGGAGAT AAACGCCGTC TCCTATCGTA TATCCTCTCT GGCACTAACT ATCTTCAATCTTAAAGGCTC CATCCCTCCA GTGACAGAGG AACACTGGGA ACGTATCAGA TCGGGACTTTTCAAACAAGT GCGGCAAGCT CAAGAGTGCT TCATGGACGA GGAGAAAGAG AACAGGGAACATCCTCACTC CGAGGACTTC CTGACAGTCT ACCTGGAGTT GGGCAAGTAT TTCTTCAGAATCAAAAAGTT CCTGATAAAT AAGAAATACA GTTTCTGTGC ATGGAAGATT GTCACAGTGGAAATAAGAAG ATGTTTCATT ATATTTTCCA AGTCCAGAAA ACTACTCAAA ATGATATCAGAATCACCCAC CTTCAAGCAA GAACTTAAAT AGAAGCTGCA ATTGCTCAAA TGTCTCCAAGAACGCTTTAT TCTAAAGCCA TTACCAGGAT GCTGCTAATG CTACCTTCAG ATGCAAGACTTTTCAAGTTC AGGGTTCAAG GCAGTGCAGT CAAAGAAAGT CTTAAGCAAA AGATGAAC

The encoded polypeptide has the following amino acid sequence:MTPKFLWLVA LVALYIPPIQ SLNCVYLDDS ILENVKLLGS TMTGFPLRCL KDITDFKFPKEILPYIQHMK REINAVSYRI SSLALTIFNL KGSIPPVTEE HWERIRSGLF KQVRQAQECFMDEEKENREH PHSEDFLTVY LELGKYFFRI KKFLINKKYS FCAWKIVTVE IRRCFIIFSKSRKLLKMISE SPTFKQELK (SEQ ID

NO:2). The Zcyto13 form of an interferon-α gene encodes a polypeptide of199 amino acids, as shown in SEQ ID NO:2. The signal sequence forZcyto13 can be predicted as comprising Met¹ through Ser²¹ of SEQ IDNO:2. The mature peptide for Zcyto13 begins at Leu²². Additionalstructural features of Zcyto13 are summarized in Table 4.

Hybridization analyses indicate that the Zcyto13 gene is stronglyexpressed in murine heart and liver tissue, and to a lesser extent, inmurine brain, kidney, uterine, spleen, and seven-day embryo tissues.Zcyto13 RNA was also detected in lung, skeletal muscle, and testistissues, as well as in the tissues of 11-, 15-, and 17-day embryos.Typically, Zcyto13 mRNA appeared as bands of about 2.8 and 6.6kilobases. These results show that the interferon-α sequences can beused differentiate among various tissues.

Southern analyses revealed that a Zcyto13 probe bound with rat and mousegenomic DNA. When incubated with EcoRI-digested DNA, the probehybridized with fragments of 4.8 and 5.65 kilobases in murine genomicDNA, and 2.1 and 5.2 kilobases in rat genomic DNA.

The Zcyto13 gene has been mapped to mouse chromosome 4 (framework markerD4Mit94, located at 4.6 centimorgans). The murine interferon-α/-β genecluster is also located at chromosome 4.

As described below, the present invention provides isolated polypeptideshaving an amino acid sequence that is at least 70%, at least 80%, or atleast 90% identical to either amino acid residues 22 to 199 of SEQ IDNO:2 or amino acid residues 1 to 199 of SEQ ID NO:2, wherein suchisolated polypeptides can either (a) specifically bind with an antibodythat specifically binds with a polypeptide consisting of the amino acidsequence of SEQ ID NO:2, or (b) exhibits anti-viral activity oranti-proliferative activity. An illustrative polypeptide is apolypeptide that comprises a first amino acid sequence consisting ofamino acid residues 22 to 199 of SEQ ID NO:2, or a polypeptide thatfurther comprises a signal secretory sequence that resides in anamino-terminal position relative to the first amino acid sequence,wherein the signal secretory sequence comprises amino acid residues 1 to21 of the amino acid sequence of SEQ ID NO:2.

Other exemplary polypeptides include polypeptides that comprise at leastone of the following amino acid motifs: (a) the amino acid sequence[FSPN][PFG][LK][RSI][CN]L[KT][DY][IR][TQKA]DF[GK][FI]P (SEQ ID NO:16),wherein the sequence is further defined by at least one conditionselected from the group consisting of: (i) the first residue is F, (ii)the fourth residue is R, (iii) the ninth residue is I, and (iv) thetenth residue is T, or (b) the amino acid sequence[KVR][FY]L[IRK][NEKL][KM]K[YH][SN][FPLS][CY]AW[KEM][IV][IV][TR][VA]E(SEQ ID NO:17), wherein the sequence is further defined by at least onecondition selected from group consisting of: (i) the first residue is K,(ii) the fourth residue is I, (iii) the fifth residue is N, (iv) thetenth residue is F, (v) the fourteenth residue is K, (vi) the fifteenthresidue is I, (vii) the seventeenth residue is T, and (viii) theeighteenth residue is V. Moreover, illustrative polypeptides compriseboth motif (a) and motif (b).

Additional exemplary polypeptides include polypeptides comprising anamino acid sequence of 15, 20, or 30 contiguous amino acid residues ofthe following amino acid sequences within SEQ ID NO:2: amino acidresidues 22 to 199, amino acid residues 22 to 188, amino acid residues22 to 45, amino acid residues 46 to 64, amino acid residues 65 to 89,amino acid residues 99 to 124, amino acid residues 134 to 155, aminoacid residues 22 to 89, and amino acid residues 161 to 181. Otherillustrative polypeptides comprise an amino acid sequence selected fromthe group consisting of: amino acid residues 22 to 199 of SEQ ID NO:2,amino acid residues 22 to 188 of SEQ ID NO:2, amino acid residues 22 to45 of SEQ ID NO:2 , amino acid residues 46 to 64 of SEQ ID NO:2 , aminoacid residues 65 to 89 of SEQ ID NO:2 , amino acid residues 99 to 124 ofSEQ ID NO:2, amino acid residues 134 to 155 of SEQ ID NO:2, amino acidresidues 22 to 89 of SEQ ID NO:2, and amino acid residues 161 to 181 ofSEQ ID NO:2. Additional examples include polypeptides consisting of anamino acid sequence selected from the group consisting of: amino acidresidues 22 to 199 of SEQ ID NO:2, amino acid residues 22 to 188 of SEQID NO:2, amino acid residues 22 to 45 of SEQ ID NO:2, amino acidresidues 46 to 64 of SEQ ID NO:2 , amino acid residues 65 to 89 of SEQID NO:2, amino acid residues 99 to 124 of SEQ ID NO:2, amino acidresidues 134 to 155 of SEQ ID NO:2, amino acid residues 22 to 89 of SEQID NO:2, and amino acid residues 161 to 181 of SEQ ID NO:2.

The polypeptides described herein can further comprise an affinity tag.

The present invention further provides antibodies and antibody fragmentsthat specifically bind with such polypeptides. Exemplary antibodiesinclude polyclonal antibodies, murine monoclonal antibodies, humanizedantibodies derived from murine monoclonal antibodies, and humanmonoclonal antibodies. Illustrative antibody fragments include F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, and minimal recognition units. The presentinvention also includes anti-idiotype antibodies that specifically bindwith such antibodies or antibody fragments. Certain anti-idiotypeantibodies, or anti-idiotype antibody fragments, possesses anti-viralactivity or anti-proliferative activity.

The present invention further includes compositions comprising a carrierand a peptide, polypeptide, antibody, or anti-idiotype antibodydescribed herein. For example, the composition can be a pharmaceuticalcomposition, and the carrier can be a pharmaceutically acceptablecarrier.

The present invention also provides isolated nucleic acid molecules thatencode a Zcyto13 polypeptide, wherein the nucleic acid molecule isselected from the group consisting of (a) a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:3, (b) a nucleic acidmolecule encoding the amino acid sequence of SEQ ID NO:2, and (c) anucleic acid molecule that remains hybridized following stringent washconditions to a nucleic acid molecule consisting of the nucleotidesequence of SEQ ID NO: 1, or the complement of SEQ ID NO: 1.

Illustrative nucleic acid molecules include those in which anydifference between the amino acid sequence encoded by the nucleic acidmolecule and the corresponding amino acid sequence of SEQ ID NO:2 is dueto a conservative amino acid substitution. The present invention furthercontemplates isolated nucleic acid molecules that comprise a nucleotidesequence of nucleotides 86 to 619 of SEQ ID NO:1.

The present invention also includes vectors and expression vectorscomprising such nucleic acid molecules. Such expression vectors maycomprise a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator. The present invention further includesrecombinant host cells comprising these vectors and expression vectors.Illustrative host cells include bacterial, yeast, fungal, avian, insect,mammalian, and plant cells. Recombinant host cells comprising suchexpression vectors can be used to prepare Zcyto13 polypeptides byculturing such recombinant host cells that comprise the expressionvector and that produce the Zcyto13 protein, and, optionally, isolatingthe Zcyto13 protein from the cultured recombinant host cells. Inaddition, the present invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and at least one ofsuch an expression vector or recombinant virus comprising suchexpression vectors.

The present invention also contemplates methods for detecting thepresence of Zcyto13 RNA in a biological sample, comprising the steps of(a) contacting a Zcyto13 nucleic acid probe under hybridizing conditionswith either (i) test RNA molecules isolated from the biological sample,or (ii) nucleic acid molecules synthesized from the isolated RNAmolecules, wherein the probe has a nucleotide sequence comprising aportion of the nucleotide sequence of SEQ ID NO:1, or its complement,and (b) detecting the formation of hybrids of the nucleic acid probe andeither the test RNA molecules or the synthesized nucleic acid molecules,wherein the presence of the hybrids indicates the presence of Zcyto13RNA in the biological sample.

The present invention further provides methods for detecting thepresence of Zcyto13 polypeptide in a biological sample, comprising thesteps of: (a) contacting the biological sample with an antibody or anantibody fragment that specifically binds with a polypeptide consistingof the amino acid sequence of SEQ ID NO:2, wherein the contacting isperformed under conditions that allow the binding of the antibody orantibody fragment to the biological sample, and (b) detecting any of thebound antibody or bound antibody fragment. Such an antibody or antibodyfragment may further comprise a detectable label selected from the groupconsisting of radioisotope, fluorescent label, chemiluminescent label,enzyme label, bioluminescent label, and colloidal gold.

The present invention also provides kits for performing these detectionmethods. For example, a kit for detection of Zcyto13 gene expression maycomprise a container that comprises a nucleic acid molecule, wherein thenucleic acid molecule is selected from the group consisting of (a) anucleic acid molecule comprising the nucleotide sequence of nucleotides86 to 619 of SEQ ID NO:1, (b) a nucleic acid molecule comprising thecomplement of nucleotides 86 to 619 of the nucleotide sequence of SEQ IDNO:1, (c) a nucleic acid molecule that is a fragment of (a) consistingof at least eight nucleotides, and (d) a nucleic acid molecule that is afragment of (b) consisting of at least eight nucleotides. Such a kit mayalso comprise a second container that comprises one or more reagentscapable of indicating the presence of the nucleic acid molecule. On theother hand, a kit for detection of Zcyto13 protein may comprise acontainer that comprises an antibody, or an antibody fragment, thatspecifically binds with a polypeptide consisting of the amino acidsequence of SEQ ID NO:2.

The present invention also contemplates anti-idiotype antibodies, oranti-idiotype antibody fragments, that specifically bind an antibody orantibody fragment that specifically binds a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:2, wherein the anti-idiotypeantibody, or anti-idiotype antibody fragment, possesses anti-viralactivity or anti-proliferative activity.

The present invention also provides isolated nucleic acid moleculescomprising a nucleotide sequence that encodes a Zcyto13 secretion signalsequence and a nucleotide sequence that encodes a biologically activepolypeptide, wherein the Zcyto13 secretion signal sequence comprises anamino acid sequence of residues 1 to 21 of SEQ ID NO:2. Illustrativebiologically active polypeptides include Factor VIIa, proinsulin,insulin, follicle stimulating hormone, tissue type plasminogenactivator, tumor necrosis factor, interleukin, colony stimulatingfactor, interferon, erythropoietin, and thrombopoietin. Moreover, thepresent invention provides fusion proteins comprising a Zcyto13secretion signal sequence and a polypeptide, wherein the Zcyto13secretion signal sequence comprises an amino acid sequence of residues 1to 21 of SEQ ID NO:2. Additional fusion proteins comprise a Zcyto13moiety and an immunoglobulin moiety. An illustrative immunoglobulinmoiety is an immunoglobulin heavy chain constant region, such as a humanF_(c) fragment. The present invention also includes isolated nucleicacid molecules that encode such fusion proteins.

The present invention further contemplates variant Zcyto13 polypeptides,wherein the arnino acid sequence of the variant is characterized by atleast one amino acid substitution within SEQ ID NO:2 selected from thegroup consisting of: (a) an alanine for glycine³⁹, (b) a valine forleucine⁶³, (c) a threonine for serine⁸¹, (d) a valine for isoleucine¹⁰⁵,and (e) a leucine for valine¹¹³. Other variant Zcyto13 polypeptides havean amino acid sequence that shares an identity with the amino acidsequence of SEQ ID NO:2 selected from the group consisting of at least70% identity, at least 80% identity, at least 90% identity, at least 95%identity, or greater than 95% identity, and wherein any differencebetween the amino acid sequence of the variant polypeptide and the aminoacid sequence of SEQ ID NO:2 is due to one or more conservative aminoacid substitutions. The present invention also includes isolatedpolypeptides consisting of an amino acid sequence of amino acid residues22-89 of SEQ ID NO:2.

The present invention further includes methods of inhibiting a viralinfection of cells, comprising the step of administering a compositioncomprising Zcyto13 to the cells. For example, the composition can be apharmaceutical composition, which is administered in a therapeuticallyeffective amount to a subject, which has a viral infection. In vivotreatment of a viral infection can provide at least one physiologicaleffect selected from the group consisting of decreased viral titer,decreased detectable viral antigen, and increased anti-viral antibodytiter.

The present invention also includes methods of inhibiting theproliferation of tumor cells, comprising the step of administering acomposition comprising Zcyto13 to the tumor cells. In an in vivoapproach, the composition is a pharmaceutical composition, administeredin a therapeutically effective amount to a subject, which has a tumor.Such in vivo administration can provide at least one physiologicaleffect selected from the group consisting of decreased number of tumorcells, decreased metastasis, decreased size of a solid tumor, andincreased necrosis of a tumor.

The present invention also provides fusion proteins comprising a Zcyto13polypeptide moiety. Such fusion proteins can further comprise animmunoglobulin moiety. An exemplary immunoglobulin moiety is a humanimmunoglobulin heavy chain constant region.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ (SEQ ID NO:18) is complementary to 5′CCCGTGCAT 3′ (SEQ ID NO:19).

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule. For example, representativecontigs to the polynucleotide sequence 5′ ATGGAGCTT 3′ (SEQ ID NO:20)are 5′ AGCTTgagt 3′ (SEQ ID NO:21) and 3′ tcgacTACC 5′ (SEQ ID NO:22).

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRF,/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner. For example, theZcyto13 regulatory element preferentially induces gene expression inheart, liver, brain, kidney, and spleen tissues, as opposed to lung,skeletal muscle, and testis tissues.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces theZcyto13 form of interferon-α from an expression vector. In contrast,Zcyto13 can be produced by a cell that is a “natural source” of Zcyto13,and that lacks an expression vector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a Zcyto13polypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of Zcyto13using affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of an anti-Zcyto13antibody, and thus, an anti-idiotype antibody mimics an epitope ofZcyto13.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-Zcyto13 monoclonal antibody fragmentbinds with an epitope of Zcyto13.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom, which can be conjugated toan antibody moiety to produce a molecule useful for diagnosis. Examplesof detectable labels include chelators, photoactive agents,radioisotopes, fluorescent agents, paramagnetic ions, or other markermoieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNAs encoding affinity tags are available fromcommercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

An “immunoconjugate” is a conjugate of an antibody component with atherapeutic agent or a detectable label.

As used herein, the term “antibody fusion protein” refers to arecombinant molecule that comprises an antibody component and atherapeutic agent. Examples of therapeutic agents suitable for suchfusion proteins include immunomodulators (“antibody-immunomodulatorfusion protein”) and toxins (“antibody-toxin fusion protein”).

A “tumor associated antigen” is a protein normally not expressed, orexpressed at lower levels, by a normal counterpart cell. Examples oftumor associated antigens include alpha-fetoprotein, carcinoembryonicantigen, and Her-2/neu. Many other illustrations of tumor associatedantigens are known to those of skill in the art. See, for example, Urbanet al., Ann. Rev. Immunol. 10:617 (1992).

As used herein, an “infectious agent” denotes both microbes andparasites. A “microbe” includes viruses, bacteria, rickettsia,mycoplasma, protozoa, fungi and like microorganisms. A “parasite”denotes infectious, generally microscopic or very small multicellularinvertebrates, or ova or juvenile forms thereof, which are susceptibleto immune-mediated clearance or lytic or phagocytic destruction, such asmalarial parasites, spirochetes, and the like.

An “infectious agent antigen” is an antigen associated with aninfectious agent.

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

An “antigenic peptide” is a peptide, which will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex whichis recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for Zcyto13” or a “Zcyto13anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the Zcyto13 gene,or (b) capable of forming a stable duplex with a portion of an mRNAtranscript of the Zcyto13 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant murine Zcyto13 gene” refers to nucleic acid moleculesthat encode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:2. Such variants include naturally-occurringpolymorphisms of Zcyto13 genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:2. Additional variant forms of Zcyto13 genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant Zcyto13 gene can be identified bydetermining whether the gene hybridizes with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1, or its complement, understringent conditions.

Alternatively, variant Zcyto13 genes can be identified by sequencecomparison. Two amino acid sequences have “100% amino acid sequenceidentity” if the amino acid residues of the two amino acid sequences arethe same when aligned for maximal correspondence. Similarly, twonucleotide sequences have “100% nucleotide sequence identity” if thenucleotide residues of the two nucleotide sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinformatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing two nucleotide or aminoacid sequences by determining optimal alignment are well-known to thoseof skill in the art (see, for example, Peruski and Peruski, The Internetand the New Biology: Tools for Genomic and Molecular Research (ASMPress, Inc. 1997), Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

Regardless of the particular method used to identify a variant Zcyto13gene or variant Zcyto13 polypeptide, a variant gene or polypeptideencoded by a variant gene is functionally characterized by either itsanti-viral or anti-proliferative activities, or by the ability to bindspecifically to an anti-Zcyto13 antibody.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The present invention includes functional fragments of Zcyto13 genes.Within the context of this invention, a “functional fragment” of aZcyto13 gene refers to a nucleic acid molecule that encodes a portion ofa Zcyto13 polypeptide, which either (1) possesses an anti-viral oranti-proliferative activity, or (2) specifically binds with ananti-Zcyto13 antibody. For example, a functional fragment of a Zcyto13gene comprises a portion of the nucleotide sequence of SEQ ID NO:1, andencodes a polypeptide having either an anti-viral or anti-proliferativeactivity.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

3. Production of the Zcyto13 Gene

Nucleic acid molecules encoding a murine Zcyto13 gene can be obtained byscreening a murine cDNA or genomic library using polynucleotide probesbased upon SEQ ID NO:1. These techniques are standard andwell-established.

As an illustration, a nucleic acid molecule that encodes a murineZcyto13 gene can be isolated from a murine cDNA library. In this case,the first step would be to prepare the cDNA library by isolating RNAfrom a tissue, such as uterine, heart, or liver tissue, using methodswell-known to those of skill in the art. In general, RNA isolationtechniques must provide a method for breaking cells, a means ofinhibiting RNase-directed degradation of RNA, and a method of separatingRNA from DNA, protein, and polysaccharide contaminants. For example,total RNA can be isolated by freezing tissue in liquid nitrogen,grinding the frozen tissue with a mortar and pestle to lyse the cells,extracting the ground tissue with a solution of phenol/chloroform toremove proteins, and separating RNA from the remaining impurities byselective precipitation with lithium chloride (see, for example, Ausubelet al. (eds.), Short Protocols in Molecular Biology, 3rd Edition, pages4-1 to 4-6 (J.ohn Wiley & Sons 1995) [“Ausubel (1995)”]; Wu et al.,Methods in Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) [“Wu(1997)”]).

Alternatively, total RNA can be isolated from uterine tissue (or, hearttissue or liver tissue) by extracting ground tissue with guanidiniumisothiocyanate, extracting with organic solvents, and separating RNAfrom contaminants using differential centrifugation (see, for example,Chirgwin et al., Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1to 4-6; Wu (1997) at pages 33-41).

In order to construct a cDNA library, poly(A)⁺ RNA must be isolated froma total RNA preparation. Poly(A)⁺ RNA can be isolated from total RNAusing the standard technique of oligo(dT)-cellulose chromatography (see,for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972);Ausubel (1995) at pages 40-11 to 4-12).

Double-stranded cDNA molecules are synthesized from poly(A)⁺ RNA usingtechniques well-known to those in the art. (see, for example, Wu (1997)at pages 41-46). Moreover, commercially available kits can be used tosynthesize double-stranded cDNA molecules. For example, such kits areavailable from Life Technologies, Inc. (Gaithersburg, Md.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Promega Corporation (Madison,Wis.) and STRATAGENE (La Jolla, Calif.).

Various cloning vectors are appropriate for the construction of a cDNAlibrary. For example, a cDNA library can be prepared in a vector derivedfrom bacteriophage, such as a λgt10 vector. See, for example, Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and )λgt11,” inDNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRLPress, 1985); Wu (1997) at pages 47-52.

Alternatively, double-stranded cDNA molecules can be inserted into aplasmnid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla,Calif.), a LAMDAGEM4 (Promega Corp.) or other commercially availablevectors. Suitable cloning vectors also can be obtained from the AmericanType Culture Collection (Manassas, Va.).

To amplify the cloned CDNA molecules, the cDNA library is inserted intoa prokaryotic host, using standard techniques. For example, a cDNAlibrary can be introduced into competent E. coli DH5 cells, which can beobtained, for example, from Life Technologies, Inc. (Gaithersburg, Md.).

A murine genomic library can be prepared by means well-known in the art(see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) atpages 307-327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic librarycan be obtained by the random shearing of genomic DNA or by the partialdigestion of genomic DNA with restriction endonucleases. Genomic DNAfragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well-known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).

Nucleic acid molecules that encode a murine Zcyto13 gene can also beobtained using the polymerase chain reaction (PCR) with oligonucleotideprimers having nucleotide sequences that are based upon the nucleotidesequences of the Zcyto13 gene, as described herein. General methods forscreening libraries with PCR are provided by, for example, Yu et al.,“Use of the Polymerase Chain Reaction to Screen Phage Libraries,” inMethods in Molecular Biology, Vol. 15: PCR Protocols: Current Methodsand Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).Moreover, techniques for using PCR to isolate related genes aredescribed by, for example, Preston, “Use of Degenerate OligonucleotidePrimers and the Polymerase Chain Reaction to Clone Gene Family Members,”in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methodsand Applications, White (ed.), pages 317-337 (Humana Press, Inc. 1993).

A library containing cDNA or genomic clones can be screened with one ormore polynucleotide probes based upon SEQ ID NO:1, using standardmethods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).

Anti-Zcyto13 antibodies, produced as described below, can also be usedto isolate DNA sequences that encode murine Zcyto13 genes from cDNAlibraries. For example, the antibodies can be used to screen λgt11expression libraries, or the antibodies can be used for immunoscreeningfollowing hybrid selection and translation (see, for example, Ausubel(1995) at pages 6-12 to 6-16; Margolis et al., “Screening λ expressionlibraries with antibody and protein probes,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), pages 1-14(Oxford University Press 1995)).

As an alternative, a Zcyto13 gene can be obtained by synthesizingnucleic acid molecules using mutually priming long oligonucleotides andthe nucleotide sequences described herein (see, for example, Ausubel(1995) at pages 8-8 to 8-9). Established techniques using the polymerasechain reaction provide the ability to synthesize DNA molecules at leasttwo kilobases in length (Adang et al., Plant Molec. Biol. 21:1131(1993), Bambot et al., PCR Methods and Applications 2:266 (1993), Dillonet al., “Use of the Polymerase Chain Reaction for the Rapid Constructionof Synthetic Genes,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 263-268,(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.4:299 (1995)).

The nucleic acid molecules of the present invention can also besynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically-synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes (>300 base pairs), however, special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length. For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994), Itakura et al., Annu. Rev. Biochem. 53:323 (1984), andClimie et al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

The sequence of a Zcyto13 cDNA or Zcyto13 genomic fragment can bedetermined using standard methods. Zcyto13 polynucleotide sequencesdisclosed herein can also be used as probes or primers to clone 5′non-coding regions of a Zcyto13 gene. In view of the tissue-specificexpression observed for Zcyto13 by northern blotting, this gene regionis expected to provide for preferential expression in heart, liver,brain, kidney, and spleen tissues. Promoter elements from a Zcyto13 genecould thus be used to direct the tissue-specific expression ofheterologous genes in, for example, transgenic animals or patientstreated with gene therapy. The identification of genomic fragmentscontaining a Zcyto13 promoter or regulatory element can be achievedusing well-established techniques, such as deletion analysis (see,generally, Ausubel (1995)).

Cloning of 5′ flanking sequences also facilitates production of Zcyto13proteins by “gene activation,” as disclosed in U.S. Pat. No. 5,641,670.Briefly, expression of an endogenous Zcyto13 gene in a cell is alteredby introducing into the Zcyto13 locus a DNA construct comprising atleast a targeting sequence, a regulatory sequence, an exon, and anunpaired splice donor site. The targeting sequence is a Zcyto13 5′non-coding sequence that permits homologous recombination of theconstruct with the endogenous Zcyto13 locus, whereby the sequenceswithin the construct become operably linked with the endogenous Zcyto13coding sequence. In this way, an endogenous Zcyto13 promoter can bereplaced or supplemented with other regulatory sequences to provideenhanced, tissue-specific, or otherwise regulated expression.

4. Production of Zcyto13 Gene Variants

The present invention provides a variety of nucleic acid molecules,including DNA and RNA molecules, which encode the Zcyto13 polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NO:3is a degenerate nucleotide sequence that encompasses all nucleic acidmolecules that encode the Zcyto13 polypeptide of SEQ ID NO:2. Thoseskilled in the art will recognize that the degenerate sequence of SEQ IDNO:3 also provides all RNA sequences encoding SEQ ID NO:2, bysubstituting U for T. Thus, the present invention contemplates Zcyto13polypeptide-encoding nucleic acid molecules comprising nucleotide 86 tonucleotide 619 of SEQ ID NO:1, and their RNA equivalents.

Table 1 sets forth the one-letter codes used within SEQ ID NO:3 todenote degenerate nucleotide positions. “Resolutions” are thenucleotides denoted by a code letter. “Complement” indicates the codefor the complementary nucleotide(s). For example, the code Y denoteseither C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:3, encompassing all possiblecodons for a given amino acid, are set forth in Table 2.

TABLE 2 One Letter Degenerate Amino Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn/Asp B RAY Glu/Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding an amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances., encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

Different species can exhibit “preferential codon usage.” In general,see, Grantham et al., Nuc. Acids Res. 8:1893 (1980), Haas et al. Curr.Biol. 6:315 (1996), Wain-Hobson et al, Gene 13:355 (1981), Grosjean andFiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986),Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin.Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995),and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 2). Forexample, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequence disclosed in SEQ ID NO:3 serves as a templatefor optimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein. Sequencescontaining preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

The present invention further provides variant polypeptides and nucleicacid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. Of particular interest are Zcyto13 polypeptidesfrom other mammalian species, including human, porcine, ovine, bovine,canine, feline, equine, and other primate polypeptides. Orthologs ofmurine Zcyto13 can be cloned using information and compositions providedby the present invention in combination with conventional cloningtechniques. For example, a Zcyto13 cDNA can be cloned using mRNAobtained from a tissue or cell type that expresses Zcyto13 as disclosedherein. Suitable sources of mRNA can be identified by probing northernblots with probes designed from the sequences disclosed herein. Alibrary is then prepared from mRNA of a positive tissue or cell line.

A Zcyto13-encoding cDNA can be isolated by a variety of methods, such asby probing with a complete or partial murine cDNA or with one or moresets of degenerate probes based on the disclosed sequences. A cDNA canalso be cloned using the polymerase chain reaction with primers designedfrom the representative murine Zcyto13 sequences disclosed herein.

In addition, a cDNA library can be used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to Zcyto13 polypeptide. Kawade et al., Antiviral Res. 1:167(1981), have shown that α types of human and mouse interferon share anantigenic homology. Accordingly, anti-Zycto13 antibodies can be used toisolate human Zcyto13 from natural sources, and to isolate human Zcyto13sequences from a library that expresses cloned DNA.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of murine Zcyto13, and thatallelic variation and alternative splicing are expected to occur.Allelic variants of this sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the nucleotide sequence shown in SEQ IDNO:1, including those containing silent mutations and those in whichmutations result in amino acid sequence changes, are within the scope ofthe present invention, as are proteins which are allelic variants of SEQID NO:2. cDNA molecules generated from alternatively spliced mRNAs,which retain the properties of the Zcyto13 polypeptide are includedwithin the scope of the present invention, as are polypeptides encodedby such cDNAs and mRNAs. Allelic variants and splice variants of thesesequences can be cloned by probing cDNA or genomic libraries fromdifferent individuals or tissues according to standard procedures knownin the art.

Within certain embodiments of the invention, isolated nucleic acidmolecules that encode murine Zcyto13 can hybridize to nucleic acidmolecules having the nucleotide sequence of SEQ ID NO:1, or a sequencecomplementary thereto, under “stringent conditions.” In general,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe.

As an illustration, a nucleic acid molecule encoding a variant Zcyto13polypeptide can be hybridized with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) at 42° C.overnight in a solution comprising 50% formamide, 5×SSC (1×SSC: 0.15 Msodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution (100×Denhardt's solution: 2% (w/v) Ficoll400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin),10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.One of skill in the art can devise variations of these hybridizationconditions. For example, the hybridization mixture can be incubated at ahigher temperature, such as about 65° C., in a solution that does notcontain formamide.

Moreover, premixed hybridization solutions are available (e.g,EXPRESSHYB Hybridization Solution from CLONTECH Laboratories, Inc.), andhybridization can be performed according to the manufacturer'sinstructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. That is, nucleic acid moleculesencoding a variant Zcyto13 polypeptide remain hybridized with a nucleicacid molecule consisting of the nucleotide sequence of SEQ ID NO:1 (orits complement) following stringent washing conditions, in which thewash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C.,including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, forexample, by substituting SSPE for SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variantZcyto13 polypeptide remain hybridized with a nucleic acid moleculeconsisting of the nucleotide sequence of SEQ ID NO:1 (or its complement)following highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.,including 0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at65° C.

The present invention also provides isolated Zcyto13 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides having at least70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the sequences shown in SEQ ID NO:2, or theirorthologs.

The present invention also contemplates Zcyto13 variant nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NO:2, and a hybridization assay, as described above.Such Zcyto13 variants include nucleic acid molecules (1) that remainhybridized with a nucleic acid molecule consisting of the nucleotidesequence of nucleotides 86 to 619 of SEQ ID NO:1 (or its complement)following stringent washing conditions, in which the wash stringency isequivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C., and (2) that encodea polypeptide having at least 70%, at least 80%, at least 90%, at least95% or greater than 95% sequence identity to the amino acid sequence ofSEQ ID NO:2. Alternatively, certain Zcyto13 variants can becharacterized as nucleic acid molecules (1) that remain hybridized witha nucleic acid molecule consisting of the nucleotide sequence ofnucleotides 86 to 619 of SEQ ID NO:1 (or its complement) followinghighly stringent washing conditions, in which the wash stringency isequivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and (2) thatencode a polypeptide having at least 70%, at least 80%, at least 90%, atleast 95% or greater than 95% sequence identity to the amino acidsequence of SEQ ID NO:2.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100).

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A  4 R −1  5 N −2  0  6D −2 −2  1  6 C  0 −3 −3 −3  9 Q −1  1  0  0 −3  5 E −1  0  0  2 −4  2 5 G  0 −2  0 −1 −3 −2 −2  6 H −2  0  1 −1 −3  0  0 −2  8 I −1 −3 −3 −3−1 −3 −3 −4 −3  4 L −1 −2 −3 −4 −1 −2 −3 −4 −3  2  4 K −1  2  0 −1 −3  1 1 −2 −1 −3 −2  5 M −1 −1 −2 −3 −1  0 −2 −3 −2  1  2 −1  5 F −2 −3 −3 −3−2 −3 −3 −3 −1  0  0 −3  0  6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2−4  7 S  1 −1  1  0 −1  0  0  0 −1 −2 −2  0 −1 −2 −1  4 T  0 −1  0 −1 −1−1 −1 −2 −2 −1 −1 −1 −1 −2 −1  1  5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2−3 −1  1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3  2 −1 −1 −2 −1  3 −3 −2−2  2  7 V  0 −3 −3 −3 −1 −2 −2 −3 −3  3  1 −2  1 −1 −2 −2  0 −3 −1  4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativeZcyto13 variant. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequencesimilarity by identifying regions shared by the query sequence (e.g.,SEQ ID NO:2) and a test sequence that have either the highest density ofidentities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM. J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom four to six.

The present invention includes nucleic acid molecules that encode apolypeptide having a conservative amino acid change, compared with theamino acid sequence of SEQ ID NO:2. That is, variants can be obtainedthat contain one or more amino acid substitutions of SEQ ID NO:2, inwhich an alkyl amino acid is substituted for an alkyl amino acid in aZcyto13 amino acid sequence, an aromatic amino acid is substituted foran aromatic amino acid in a Zcyto13 amino acid sequence, asulfur-containing amino acid is substituted for a sulfur-containingamino acid in a Zcyto13 amino acid sequence, a hydroxy-containing aminoacid is substituted for a hydroxy-containing amino acid in a Zcyto13amino acid sequence, an acidic amino acid is substituted for an acidicamino acid in a Zcyto13 amino acid sequence, a basic amino acid issubstituted for a basic amino acid in a Zcyto13 amino acid sequence, ora dibasic monocarboxylic amino acid is substituted for a dibasicmonocarboxylic amino acid in a Zcyto13 amino acid sequence.

Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine. For example, variant Zcyto13polypeptides that have an amino acid sequence that differs from SEQ IDNO:2 can be obtained by substituting alanine for glycine³⁹, valine forleucine⁶³, threonine for serine⁸¹, valine for isoleucine¹⁰⁵, or leucinefor valine¹¹³. Additional variants can be obtained by producingpolypeptides having two or more of these amino acid substitutions.

The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915(1992)). Accordingly, the BLOSUM62 substitution frequencies can be usedto define conservative amino acid substitutions that may be introducedinto the amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” can refer to a substitution represented by a BLOSUM62value of greater than −1. For example, an amino acid substitution isconservative if the substitution is characterized by a BLOSUM62 value of0, 1, 2, or 3. According to this system, preferred conservative aminoacid substitutions are characterized by a BLOSUM62 value of at least 1(e.g., 1, 2 or 3), while more preferred conservative amino acidsubstitutions are characterized by a BLOSUM62 value of at least 2 (e.g.,2 or 3).

Particular variants of murine Zcyto13 are characterized by having atleast 70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the corresponding amino acid sequence (Le., SEQ IDNO:2), wherein the variation in amino acid sequence is due to one ormore conservative amino acid substitutions.

Conservative amino acid changes in a Zcyto13 gene can be introduced bysubstituting nucleotides for the nucleotides recited in SEQ ID NO:1.Such “conservative amino acid” variants can be obtained, for example, byoligonucleotide-directed mutagenesis, linker-scanning mutagenesis,mutagenesis using the polymerase chain reaction, and the like (seeAusubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), DirectedMutagenesis: A Practical Approach (IRL Press 1991)). The ability of suchvariants to promote anti-viral or anti-proliferative activity can bedetermined using a standard method, such as the assay described herein.Alternatively, a variant Zcyto13 polypeptide can be identified by theability to specifically bind anti-Zcyto13 antibodies.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for Zcyto13 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'l Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., J.Biol. Chem. 271:4699 (1996). The identities of essential amino acids canalso be inferred from analysis of homologies with interferon-α,interferon-β, interferon-γ, interferon-δ, interferon-ω, andinterferon-τ, and particularly, by analysis of the murine interferon-αfamily members.

Sequence analysis can also identify motifs that reside within murineinterferon polypeptides. As an illustration, the following murineinterferon sequences were analyzed for common amino acid sequencemotifs: nine murine interferon-α polypeptides, interferon-β,interferon-δ, and Zcyto13. The results of this analysis revealed twomotifs in the interferon sequences. One motif occurs at amino acidresidues 45 to 59 of Zcyto13, and has the following sequence:[FSPN][PFG][LK][RSI][CN]L[KT][DY][IR][TQKA]DF[GK][FI]P, SEQ ID NO:16,wherein acceptable amino acids for a given position are indicated withinsquare brackets (“murine interferon motif 1”). Zcyto13 can bedistinguished from other murine interferons when the motif is furtherdefined by the following conditions: (1) the first residue of thesequence is F, or (2) the fourth residue is R, or (3) the ninth residueis I, or (4) the tenth residue is T. Accordingly, the present inventionincludes polypeptides that comprise an amino acid motif having thesequence [FSPN][PFG][LK][RSI][CN]L[KT][DY][IR][TQKA]DF[GK][FI]P, SEQ IDNO:16, wherein the sequence is further defined by at least one of thefollowing conditions: the first residue is F, the fourth residue is R,the ninth residue is I, or the tenth residue is T.

A second motif occurs at amino acid residues 152 to 170 of Zcyto13, andhas the following amino acid sequence:[KVR][FY]L[IRK][NEKL][KM]K[YH][SN][FPLS][CY]AW[KEM][IV][IV][TR][VA]E,SEQ ID NO:17, wherein acceptable amino acids for a given position areindicated within square brackets (“murine interferon motif 2”). Zcyto13can be distinguished from other murine interferons when the motif isfurther defined by one of the following conditions: (1) the firstresidue is K, or (2) the fourth residue is I or (3) the fifth residue isN, or (4) the tenth residue is F, or (5) the fourteenth residue is K, or(6) the fifteenth residue is I, or (7) the seventeenth residue is T, or(8) the eighteenth residue is V. Thus, the present invention includespolypeptides that comprise an amino acid motif having the sequence:[KVR][FY]L[IRK][NEKL][KM]K[YH][SN][FPLS][CY]AW[KEM][IV][IV][TR][VA]E,SEQ ID NO:17, wherein the sequence is further defined by at least one ofthe following conditions: the first residue is K, the fourth residue isI, the fifth residue is N, the tenth residue is F, the fourteenthresidue is K, the fifteenth residue is I, the seventeenth residue is T.or the eighteenth residue is V. The present invention also includespolypeptides having both murine interferon motifs 1 and 2.

Although sequence analysis can be used to identify Zcyto13 receptorbinding sites, the location of Zcyto13 receptor binding domains can alsobe determined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899 (1992), andWlodaver et al., FEBS Lett. 309:59 (1992). Moreover, Zcyto13 labeledwith biotin or FITC can be used for expression cloning of Zcyto13receptors.

To date, studies have identified two main receptor binding sites in TypeI interferons: one at a high affinity responsible for the binding to thereceptor, and another site at lower affinity involved in mediatingsignal transduction (see, for example, Viscomi, Biotherapy 10:59(1997)). The first site engages Helices A and B, and Loop AB, while thesecond site engages Helices A and C and Loop DE. Accordingly, mutationscan be introduced into Helix C or Loop DE to interfere with Zcyto13receptor signaling. Such a mutant would be expected to bind a Zcyto13receptor without producing a biological effect, and therefore, wouldhave the properties of a Zcyto13 antagonist. As shown in Table 4, HelixC and Loop DE are represented by amino acids 99 to 124, and 156 to 160,respectively, of SEQ ID NO:2. Another form of Zcyto13 antagonist couldconsist of Helices A and B, and Loop AB of the Zcyto13 form describedherein (ie., amino acids 22 to 89 of SEQ ID NO:2).

TABLE 4 Structural Feature Amino Acid Residues Nucleotides of of Zcyto13of SEQ ID NO:2 SEQ ID NO:1 Signal sequence  1-21 23-85 Helix A 22-45 86-157 AB Loop 46-64 158-214 Helix B 65-89 215-289 BC Loop 90-98290-316 Helix C  99-124 317-394 CD Loop 125-133 395-421 Helix D 134-155422-487 DE Loop 156-160 488-502 Helix E 161-188 503-586

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)).

Variants of the disclosed Zcyto13 nucleotide and polypeptide sequencescan also be generated through DNA shuffling as disclosed by Stemmer,Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA 91:10747(1994), and international publication No. WO 97/20078. Briefly, variantDNAs are generated by in vitro homologous recombination by randomfragmentation of a parent DNA followed by reassembly using PCR,resulting in randomly introduced point mutations. This technique can bemodified by using a family of parent DNAs, such as allelic variants orDNAs from different species, to introduce additional variability intothe process. Selection or screening for the desired activity, followedby additional iterations of mutagenesis and assay provides for rapid“evolution” of sequences by selecting for desirable mutations whilesimultaneously selecting against detrimental changes.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-Zcyto13 antibodies, can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The present invention also includes “functional fragments” of Zcyto13polypeptides and nucleic acid molecules encoding such functionalfragments. Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes a Zcyto13 polypeptide. As an illustration, DNA molecules havingthe nucleotide sequence of SEQ ID NO:1 can be digested with Bal31nuclease to obtain a series of nested deletions. The fragments are theninserted into expression vectors in proper reading frame, and theexpressed polypeptides are isolated and tested for anti-viral oranti-proliferative activity, or for the ability to bind anti-Zcyto13antibodies. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired fragment. Alternatively,particular fragments of a Zcyto13 gene can be synthesized using thepolymerase chain reaction.

Studies on the truncation at either or both termini of interferons havebeen summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507(1995). Moreover, standard techniques for functional analysis ofproteins are described by, for example, Treuter et al., Molec. GenGenet. 240:113 (1993), Content et al., “Expression and preliminarydeletion analysis of the 42 kDa 2-5A synthetase induced by humaninterferon,” in Biological Interferon Systems, Proceedings of ISIR-TNOMeeting on Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff1987), Herschman, “The EGF Receptor,” in Control of Animal CellProliferation, Vol. 1, Boynton et al., (eds.) pages 169-199 (AcademicPress 1985), Coumailleau et al., J. Biol. Chem. 270:29270 (1995);Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al.,Biochem. Pharmacol. 50:1295 (1995), and Meisel et al., Plant Molec.Biol. 30:1 (1996).

The present invention also contemplates functional fragments of aZcyto13 gene that has amino acid changes, compared with the amino acidsequence of SEQ ID NO:2. A variant Zcyto13 gene can be identified on thebasis of structure by determining the level of identity with nucleotideand amino acid sequence of SEQ ID NO:2, as discussed above. Analternative approach to identifying a variant gene on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant Zcyto13 gene can hybridize to a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1, as discussed above.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a Zcyto13 polypeptide describedherein. Such fragments or peptides may comprise an “immunogenicepitope,” which is a part of a protein that elicits an antibody responsewhen the entire protein is used as an immunogen. Immunogenicepitope-bearing peptides can be identified using standard methods (see,for example, Geysen et al., Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et at, Science 219:660 (1983)).Accordingly, antigenic epitope-bearing peptides and polypeptides of thepresent invention are usefuil to raise antibodies that bind with thepolypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides can contain at leastfour to ten amino acids, at least ten to fifteen amino acids, or about15 to about 30 amino acids of SEQ ID NO:2. Such epitope-bearing peptidesand polypeptides can be produced by fragmenting a Zcyto13 polypeptide,or by chemical peptide synthesis, as described herein. Moreover,epitopes can be selected by phage display of random peptide libraries(see, for example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993),and Cortese et al., Curr. Opin. Biotechnol. 7:616 (1996)). Standardmethods for identifying epitopes and producing antibodies from smallpeptides that comprise an epitope are described, for example, by Mole,“Epitope Mapping,” in Methods in Molecular Biology, Vol. 10, Manson(ed.), pages 105-116 (The Humana Press, Inc. 1992), Price, “Productionand Characterization of Synthetic Peptide-Derived Antibodies,” inMonoclonal Antibodies: Production, Engineering, and ClinicalApplication, Ritter and Ladyman (eds.), pages 60-84 (CambridgeUniversity Press 1995), and Coligan et al. (eds.), Current Protocols inImmunology, pages 9.3.1-9.3.5 and pages 9.4.1-9.4.11 (J.ohn Wiley & Sons1997).

Regardless of the particular nucleotide sequence of a variant Zcyto13gene, the gene encodes a polypeptide that is characterized by itsanti-viral or anti-proliferative activity, or by the ability to bindspecifically to an anti-Zcyto13 antibody. More specifically, variantmurine Zcyto13 genes encode polypeptides, which exhibit at least 50%,or, greater than 70, 80, or 90%, of the activity of polypeptide encodedby the murine Zcyto13 gene described herein.

For any Zcyto13 polypeptide, including variants and fusion proteins, oneof ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above. Moreover, those of skill in the art canuse standard software to devise Zcyto13 variants based upon thenucleotide and amino acid sequences described herein. Accordingly, thepresent invention includes a computer-readable medium encoded with adata structure that provides at least one of the following sequences:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. Suitableforms of computer-readable media include magnetic media andoptically-readable media. Examples of magnetic media include a hard orfixed drive, a random access memory (RAM) chip, a floppy disk, digitallinear tape (DLT), a disk cache, and a ZIP disk. Optically readablemedia are exemplified by compact discs (e.g., CD-read only memory (ROM),CD-rewritable (RW), and CD-recordable), and digital versatile/videodiscs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

5. Production of Zcyto13 Fusion Proteins and Conjugates

Fusion proteins of Zcyto13 can be used to express Zcyto13 in arecombinant host, and to isolate expressed Zcyto13. As described below,particular Zcyto13 fusion proteins also have uses in diagnosis andtherapy.

One type of fusion protein comprises a peptide that guides a Zcyto13polypeptide from a recombinant host cell. To direct a Zcyto13polypeptide into the secretory pathway of a eukaryotic host cell, asecretory signal sequence (also known as a signal peptide, a leadersequence, prepro sequence or pre sequence) is provided in the Zcyto13expression vector. While the secretory signal sequence may be derivedfrom Zcyto13, a suitable signal sequence may also be derived fromanother secreted protein or synthesized de novo. The secretory signalsequence is operably linked to a Zcyto13-encoding sequence such that thetwo sequences are joined in the correct reading frame and positioned todirect the newly synthesized polypeptide into the secretory pathway ofthe host cell. Secretory signal sequences are commonly positioned 5′ tothe nucleotide sequence encoding the polypeptide of interest, althoughcertain secretory signal sequences may be positioned elsewhere in thenucleotide sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Although the secretory signal sequence of Zcyto13 or another proteinproduced by mammalian cells (e.g., tissue-type plasminogen activatorsignal sequence, as described, for example, in U.S. Pat. No. 5,641,655)is useful for expression of Zcyto13 in recombinant mammalian hosts, ayeast signal sequence is preferred for expression in yeast cells.Examples of suitable yeast signal sequences are those derived from yeastmating phermone α-factor (encoded by the MFαl gene), invertase (encodedby the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene). See,for example, Romanos et al., “Expression of Cloned Genes in Yeast,” inDNA Cloning 2: A Practical Approach, 2^(nd) Edition, Glover and Hames(eds.), pages 123-167 (Oxford University Press 1995).

In bacterial cells, it is often desirable to express a heterologousprotein as a fusion protein to decrease toxicity, increase stability,and to enhance recovery of the expressed protein. For example, Zcyto13can be expressed as a fusion protein comprising a glutathioneS-transferase polypeptide. Glutathione S-transferease fusion proteinsare typically soluble, and easily purifiable from E. coli lysates onimmobilized glutathione columns. In similar approaches, a Zcyto13 fusionprotein comprising a maltose binding protein polypeptide can be isolatedwith an amylose resin column, while a fusion protein comprising theC-terminal end of a truncated Protein A gene can be purified usingIgG-Sepharose. Established techniques for expressing a heterologouspolypeptide as a fusion protein in a bacterial cell are described, forexample, by Williams et al, “Expression of Foreign Proteins in E. coliUsing Plasmid Vectors and Purification of Specific PolyclonalAntibodies,” in DNA Cloning 2: A Practical Approach, 2^(nd) Edition,Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995). Inaddition, commercially available expression systems are available. Forexample, the PINPOINT Xa protein purification system (PromegaCorporation; Madison, Wis.) provides a method for isolating a fusionprotein comprising a polypeptide that becomes biotinylated duringexpression with a resin that comprises avidin.

Peptide tags that are useful for isolating heterologous polypeptidesexpressed by either prokaryotic or eukaryotic cells includepolyHistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

The present invention also contemplates that the use of the secretorysignal sequence contained in the Zcyto13 polypeptides of the presentinvention to direct other polypeptides into the secretory pathway. Asignal fusion polypeptide can be made wherein a secretory signalsequence derived from amino acid residues 1 to 21 of SEQ ID NO:2 isoperably linked to another polypeptide using methods known in the artand disclosed herein. The secretory signal sequence contained in thefusion polypeptides of the present invention is preferably fusedamino-terminally to an additional peptide to direct the additionalpeptide into the secretory pathway. Such constructs have numerousapplications known in the art. For example, these novel secretory signalsequence fusion constructs can direct the secretion of an activecomponent of a normally non-secreted protein, such as a receptor. Suchfusions may be used in a transgenic animal or in a cultured recombinanthost to direct peptides through the secretory pathway. With regard tothe latter, exemplary polypeptides include pharmaceutically activemolecules such as Factor VIIa, proinsulin, insulin, follicle stimulatinghormone, tissue type plasminogen activator, tumor necrosis factor,interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, and IL-18), colony stimulating factors (e.g., granulocyte-colonystimulating factor (G-CSF) and granulocyte macrophage-colony stimulatingfactor (GM-CSF)), interferons (e.g., interferons-α, -β, -γ, -ω, -δ, -τ,and -ε), the stem cell growth factor designated “S1 factor,”erythropoietin, and thrombopoietin. The Zcyto13 secretory signalsequence contained in the fusion polypeptides of the present inventionis preferably fused amino-terminally to an additional peptide to directthe additional peptide into the secretory pathway. Fusion proteinscomprising a Zcyto13 secretory signal sequence can be constructed usingstandard techniques.

Another form of fusion protein comprises a Zcyto13 polypeptide and animmunoglobulin heavy chain constant region, typically an F_(c) fragment,which contains two or three constant region domains and a hinge regionbut lacks the variable region. As an illustration, Chang et al., U.S.Pat. No. 5,723,125, describe a fusion protein comprising a humaninterferon and a human immunoglobulin Fc fragment. The C-terminal of theinterferon is linked to the N-terminal of the Fc fragment by a peptidelinker moiety. An example of a peptide linker is a peptide comprisingprimarily a T cell inert sequence, which is immunologically inert. Anexemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGGS (SEQ ID NO:4). In this fusion protein, a preferred Fc moiety is ahuman γ4 chain, which is stable in solution and has little or nocomplement activating activity. Accordingly, the present inventioncontemplates a Zcyto13 fusion protein that comprises a Zcyto13 moietyand a human Fc fragment, wherein the C-terminus of the Zcyto13 moiety isattached to the N-terminus of the Fc fragment via a peptide linker, suchas a peptide consisting of the amino acid sequence of SEQ ID NO:4. TheZcyto13 moiety can be a Zcyto13 molecule or a fragment thereof.

In another variation, a Zcyto13 fusion protein comprises an IgGsequence, a Zcyto13 moiety covalently joined to the aminoterminal end ofthe IgG sequence, and a signal peptide that is covalently joined to theaminoterminal of the Zcyto13 moiety, wherein the IgG sequence consistsof the following elements in the following order: a hinge region, a CH₂domain, and a CH₃ domain. Accordingly, the IgG sequence lacks a CH₁domain. The Zcyto13 moiety displays a Zcyto13 activity, as describedherein, such as the ability to bind with a Zcyto13 receptor. Thisgeneral approach to producing fusion proteins that comprise bothantibody and nonantibody portions has been described by LaRochelle etal., EP 742830 (WO 95/21258).

Fusion proteins comprising a Zcyto13 moiety and an Fc moiety can beused, for example, as an in vitro assay tool. For example, the presenceof an Zcyto13 receptor in a biological sample can be detected using aZcyto13-immunoglobulin fusion protein, in which the Zcyto13 moiety isused to target the cognate receptor, and a macromolecule, such asProtein A or anti-Fc antibody, is used to detect the bound fusionprotein-receptor complex. Moreover, such fusion proteins can be used toidentify agonists and antagonists that interfere with the binding ofZcyto13 to its receptor.

In addition, antibody-Zcyto13 fusion proteins, comprising antibodyvariable domains, are usefuil as therapeutic proteins, in which theantibody moiety binds with a target antigen, such as a tumor associatedantigen. Methods of making antibody-cytokine fusion proteins are knownto those of skill in the art. For example, antibody fusion proteinscomprising an interleukin-2 moiety are described by Boleti et al., Ann.Oncol. 6:945 (1995), Nicolet et al., Cancer Gene Ther. 2:161 (1995),Becker et al., Proc. Nat'l Acad. Sci. USA 93:7826 (1996), Hank et al.,Clin. Cancer Res. 2:1951 (1996), and Hu et al., Cancer Res. 56:4998(1996). Moreover, Yang et al., Hum. Antibodies Hybridomas 6:129 (1995),and Xiang et al., J. Biotechnol. 53:3 (1997), describe fusion proteinsthat include an F(ab′)₂ fragment and a tumor necrosis factor alphamoiety. Additional cytokine-antibody fusion proteins include IL-8,IL-12, or interferon-τ as the cytokine moiety (Holzer et al., Cytokine8:214 (1996); Gillies et al., J. Immunol. 160:6195 (1998); Xiang et al.,Hum. Antibodies Hybridomas 7:2 (1996)). Also see, Gillies, U.S. Pat. No.5,650,150.

Moreover, using methods described in the art, hybrid Zcyto13 proteinscan be constructed using regions or domains of the inventive Zcyto13 incombination with those of other interferon family proteins (i.e.,interferon-α, interferon-β, interferon-γ, interferon-δ, interferon-ω,and interferon-τ), or heterologous proteins (see, for example, Picard,Cur. Opin. Biology 5:511 (1994)). These methods allow the determinationof the biological importance of larger domains or regions in apolypeptide of interest. Such hybrids may alter reaction kinetics,binding, constrict or expand the substrate specificity, or alter tissueand cellular localization of a polypeptide, and can be applied topolypeptides of unknown structure. For example Horisberger and DiMarco,Pharmac. Ther. 66:507 (1995), describe the construction of fusionprotein hybrids comprising different interferon-α subtypes, as well ashybrids comprising interferon-α domains from different species (alsosee, Van Heuvel et al., J. Gen. Virol. 67:2215 (1986)).

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. For example, part or all of a domain(s) conferring a biologicalfunction may be swapped between interferon-α of the present inventionwith the functionally equivalent domain(s) from another interferon-α, orfrom another family member, such as interferon-β, interferon-δ,interferon-γ, interferon-ω, or interferon-τ. Such domains include, butare not limited to, the secretory signal sequence, helices A, B, C, D,and E, and loops AB, BC, CD, and DE. Such fusion proteins would beexpected to have a biological functional profile that is the same orsimilar to polypeptides of the present invention or other knowninterferon family proteins, depending on the fusion constructed.Moreover, such fusion proteins may exhibit other properties as disclosedherein. General methods for enzymatic and chemical cleavage of fusionproteins are described, for example, by Ausubel (1995) at pages 16-19 to16-25.

The present invention also contemplates chemically modified Zcyto13compositions, in which a Zcyto13 polypeptide is linked with a polymer.Typically, the polymer is water soluble so that the Zcyto13 conjugatedoes not precipitate in an aqueous environment, such as a physiologicalenvironment. An example of a suitable polymer is one that has beenmodified to have a single reactive group, such as an active ester foracylation, or an aldehyde for alkylation. In this way, the degree ofpolymerization can be controlled. An example of a reactive aldehyde ispolyethylene glycol propionaldehyde, or mono-(C1-C10) alkoxy, or aryloxyderivatives thereof (see, for example, Harris, et al., U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. Moreover, amixture of polymers can be used to produce Zcyto13 conjugates.

Zcyto13 conjugates used for therapy can comprise pharmaceuticallyacceptable water-soluble polymer moieties. Conjugation of interferonswith water-soluble polymers has been shown to enhance the circulatinghalf-life of the interferon, and to reduce the immunogenicity of thepolypeptide (see, for example, Nieforth et al., Clin. Pharmacol. Ther.59:636 (1996), and Monkarsh et al., Anal. Biochem. 247:434 (1997)).

Suitable water-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,bis-succinimidyl carbonate PEG, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or othercarbohydrate-based polymers. Suitable PEG may have a molecular weightfrom about 600 to about 60,000, including, for example, 5,000, 12,000,20,000 and 25,000. A Zcyto13 conjugate can also comprise a mixture ofsuch water-soluble polymers.

One example of a Zcyto13 conjugate comprises a Zcyto13 moiety and apolyalkyl oxide moiety attached to the N-terminus of the Zcyto13 moiety.PEG is one suitable polyalkyl oxide. As an illustration, Zcyto13 can bemodified with PEG, a process known as “PEGylation.” PEGylation ofZcyto13 can be carried out by any of the PEGylation reactions known inthe art (see, for example, EP 0 154 316, Delgado et al., CriticalReviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan andSpreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int JHematol 68:1 (1998)). For example, PEGylation can be performed by anacylation reaction or by an alkylation reaction with a reactivepolyethylene glycol molecule. In an alternative approach, Zcyto13conjugates are formed by condensing activated PEG, in which a terminalhydroxy or amino group of PEG has been replaced by an activated linker(see, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with a Zcyto13 polypeptide. An example of an activatedPEG ester is PEG esterified to N-hydroxysuccinimide. As used herein, theterm “acylation” includes the following types of linkages betweenZcyto13 and a water soluble polymer: amide, carbanate, urethane, and thelike. Methods for preparing PEGylated Zcyto13 by acylation willtypically comprise the steps of (a) reacting a Zcyto13 polypeptide withPEG (such as a reactive ester of an aldehyde derivative of PEG) underconditions whereby one or more PEG groups attach to Zcyto13, and (b)obtaining the reaction product(s). Generally, the optimal reactionconditions for acylation reactions will be determined based upon knownparameters and desired results. For example, the larger the ratio ofPEG:Zcyto13, the greater the percentage of polyPEGylated Zcyto13product.

The product of PEGylation by acylation is typically a polyPEGylatedZcyto13 product, wherein the lysine ε-amino groups are PEGylated via anacyl linking group. An example of a connecting linkage is an amide.Typically, the resulting Zcyto13 will be at least 95% mono-, di-, ortri-pegylated, although some species with higher degrees of PEGylationmay be formed depending upon the reaction conditions. PEGylated speciescan be separated from unconjugated Zcyto13 polypeptides using standardpurification methods, such as dialysis, ultrafiltration, ion exchangechromatography, affinity chromatography, and the like.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with Zcyto13 in the presence of a reducing agent. PEGgroups are preferably attached to the polypeptide via a —CH₂—NH group.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation of Zcyto13monopolymer conjugates.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer Zcyto13 conjugate molecule can comprise the steps of: (a)reacting a Zcyto13 polypeptide with a reactive PEG under reductivealkylation conditions at a pH suitable to permit selective modificationof the α-amino group at the amino terminus of the Zcyto13, and (b)obtaining the reaction product(s). The reducing agent used for reductivealkylation should be stable in aqueous solution and preferably be ableto reduce only the Schiff base formed in the initial process ofreductive alkylation. Preferred reducing agents include sodiumborohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer Zcyto13conjugates, the reductive alkylation reaction conditions are those whichpermit the selective attachment of the water soluble polymer moiety tothe N-terminus of Zcyto13. Such reaction conditions generally providefor pKa differences between the lysine amino groups and the α-aminogroup at the N-terminus. The pH also affects the ratio of polymer toprotein to be used. In general, if the pH is lower, a larger excess ofpolymer to protein will be desired because the less reactive theN-terminal α-group, the more polymer is needed to achieve optimalconditions. If the pH is higher, the polymer:Zcyto13 need not be aslarge because more reactive groups are available. Typically, the pH willfall within the range of 3-9, or 3-6.

Another factor to consider is the molecular weight of the water-solublepolymer. Generally, the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.For PEGylation reactions, the typical molecular weight is about 2 kDa toabout 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25kDa. The molar ratio of water-soluble polymer to Zcyto13 will generallybe in the range of 1:1 to 100:1. Typically, the molar ratio ofwater-soluble polymer to Zcyto13 will be 1:1 to 20:1 for polyPEGylation,and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising interferon andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal Biochem. 247:434 (1997)).

The present invention contemplates compositions comprising a peptide orpolypeptide described herein. Such compositions can further comprise acarrier. The carrier can be a conventional organic or inorganic carrier.Examples of carriers include water, buffer solution, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, and the like.

Peptides and polypeptides of the present invention comprise at leastsix, at least nine, or at least 15 contiguous amino acid residues of thefollowing amino acid sequences within SEQ ID NO:2: amino acid residues22 to 199, amino acid residues 22 to 188, amino acid residues 22 to 45,amino acid residues 46 to 64, amino acid residues 65 to 89, amino acidresidues 90 to 98, amino acid residues 99 to 124, amino acid residues125 to 133, amino acid residues 134 to 155, amino acid residues 22 to89, and amino acid residues 161 to 181. Within certain embodiments ofthe invention, the polypeptides comprise 20, 30, 40. 50, 100, or morecontiguous residues of these amino acid sequences. Nucleic acidmolecules encoding such peptides and polypeptides are useful aspolymerase chain reaction primers and probes.

6. Production of Zcyto13 Polypeptides in Cultured Cells

The polypeptides of the present invention, including full-lengthpolypeptides, functional fragments, and fusion proteins, can be producedin recombinant host cells following conventional techniques. To expressa Zcyto13 gene, a nucleic acid molecule encoding the polypeptide must beoperably linked to regulatory sequences that control transcriptionalexpression in an expression vector and then, introduced into a hostcell. In addition to transcriptional regulatory sequences, such aspromoters and enhancers, expression vectors can include translationalregulatory sequences and a marker gene, which is suitable for selectionof cells hat carry the expression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence. As discussed above, expressionvectors can also include nucleotide sequences encoding a secretorysequence that directs the heterologous polypeptide into the secretorypathway of a host cell. For example, a Zcyto13 expression vector maycomprise a Zcyto13 gene and a secretory sequence derived from a Zcyto13gene or another secreted gene.

Zcyto13 proteins of the present invention may be expressed in mammaliancells. Examples of suitable mammalian host cells include African greenmonkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells(293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570;ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34),Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 [Chasin etal., Som. Cell. Molec. Genet. 12:555 (1986)]), rat pituitary cells (GH1;ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control Zcyto13 gene expression inmammalian cells if the prokaryotic promoter is regulated by a eukaryoticpromoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman etal., Nucl. Acids Res. 19:4485 (1991)).

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

Zcyto13 polypeptides can also be produced by cultured mammalian cellsusing a viral delivery system. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, vaccinia virus and adeno-associatedvirus (AAV). Adenovirus, a double-stranded DNA virus, is currently thebest studied gene transfer vector for delivery of heterologous nucleicacid (for a review, see Becker et al., Meth. Cell Biol. 43:161 (1994),and Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages ofthe adenovirus system include the accommodation of relatively large DNAinserts, the ability to grow to high-titer, the ability to infect abroad range of mammalian cell types, and flexibility that allows usewith a large number of available vectors containing adifferentpromoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al., Cytotechnol. 15:145 (1994)).

Zcyto13 may also be expressed in other higher eukaryotic cells, such asavian, fungal, insect, yeast, or plant cells. The baculovirus systemprovides an efficient means to introduce cloned Zcyto13 genes intoinsect cells. Suitable expression vectors are based upon the Autographacalifornica multiple nuclear polyhedrosis virus (AcMNPV), and containwell-known promoters such as Drosophila heat shock protein (hsp) 70promoter, Autographa californica nuclear polyhedrosis virusimmediate-early gene promoter (ie-1) and the delayed early 39K promoter,baculovirus p10 promoter, and the Drosophila metallothionein promoter. Asecond method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the Zcyto13 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990),Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, andRapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed Zcyto13 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952(1985)). Using a technique known in the art, a transfer vectorcontaining a Zcyto13 gene is transformed into E. coli, and screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructed,which replace the native Zcyto13 secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), orbaculovirus gp67 (PharMingen: San Diego, Calif.) can be used inconstructs to replace the native Zcyto13 secretory signal sequence.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al., “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al., “The baculovirus expression system,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 183-218 (John Wiley & Sons., Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A suitable vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A suitable selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar ptotease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

Alternatively, Zcyto13 genes can be expressed in prokaryotic host cells.Suitable promoters that can be used to express Zcyto13 polypeptides in aprokaryotic host are well-known to those of skill in the art and includepromoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, theP_(R) and P_(L) promoters of bacteriophage lambda, the trp, recA, heatshock, lacUV5, tac, lpp-lacSpr, phoA, and lacZ promoters of E. coli,promoters of B. subtilis, the promoters of the bacteriophages ofBacillus, Streptomyces promoters, the int promoter of bacteriophagelambda, the bla promoter of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene. Prokaryotic promoters have beenreviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson et al.,Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and byAusubel et al. (1995).

Illustrative prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

When expressing a Zcyto13 polypeptide in bacteria such as E. coli, thepolypeptide may be retained in the cytoplasm, typically as insolublegranules, or may be directed to the periplasmic space by a bacterialsecretion sequence. In the former case, the cells are lysed, and thegranules are recovered and denatured using, for example, guanidineisothiocyanate or urea. The denatured polypeptide can then be refoldedand dimerized by diluting the denaturant, such as by dialysis against asolution of urea and a combination of reduced and oxidized glutathione,followed by dialysis against a buffered saline solution. In the lattercase, the polypeptide can be recovered from the periplasmic space in asoluble and functional form by disrupting the cells (by, for example,sonication or osmotic shock) to release the contents of the periplasmicspace and recovering the protein, thereby obviating the need fordenaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mamrnmalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

In particular, the art of producing interferon polypeptides fromcultured cells is well-established due to the great interest ininterferon pharmaceuticals. For example, recombinant interferons havebeen produced by bacteria, yeasts, plant cells, insect cells, vertebratecells, as well as in cell-free systems (Horisberger and Di Marco,Pharmac. Ther. 66:507 (1995)). Reviews of methods for producingrecombinant interferon are provided, for example, by Edge and Camble,Biotechnol. Genet. Eng. Rev. 2:215 (1984), Langer and Pestka, J. Invest.Dermatol 83:128s (1984), Pestka, Semin. Hematol. 23:27 (1986), Baron andNarula, Crit. Rev. Biotechnol. 10:179 (1990), and Croughan et al.,Bioprocess Technol 21:377 (1995). The production of human interferon inChinese hamster ovary (CHO) cells has been described by McCormick etal., U.S. Pat. No. 5,795,779, while Dorin et al., U.S. Pat. No.5,814,485, teach methods for producing interferon in E. coli.

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al., Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205 (1998),and Kochendoerfer and Kent, Curr. Opin. Chem. Biol. 3:665 (1999)).

7. Isolation of Zcyto13 Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or even greater than 95% purity with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. The polypeptides of the presentinvention may also be purified to a pharmaceutically pure state, whichis greater than 99.9% pure. In certain preparations, a purifiedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of Zcyto13 purified from natural sources (e.g.,heart, liver, brain, kidney, or spleen), and recombinant Zcyto13polypeptides and fuision Zcyto13 polypeptides purified from recombinanthost cells. In general, ammonium sulfate precipitation and acid orchaotrope extraction may be used for fractionation of samples. Exemplarypurification steps may include hydroxyapatite, size exclusion, FPLC andreverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Phannacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sullhydryl groups, hydroxylgroups and/or carbohydrate moieties. As an illustration, Beare et al.,Biochim. Biophys. Acta 1310:81 (1996), describe the isolation of amurine interferon-α using a combination of Blue Sepharosechromatography, immunoaffinity exclusion, and Q Sepharose ion exchangefractionation.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Phannacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in Zcyto13 isolation and purification can bedevised by those of skill in the art. For example, anti-Zcyto13antibodies, obtained as described below, can be used to isolate largequantities of protein by immunoaffinity purification. The use ofmonoclonal antibody columns to purify interferons from recombinant cellsand from natural sources has been described, for example, by Staehelinet al., J. Biol. Chem. 256:9750 (1981), and by Adolf et al., J. Biol.Chem. 265:9290 (1990). Moreover, methods for binding ligands, such asZcyto13, to receptor polypeptides bound to support media are well knownin the art.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). For example, theinterferon-γ isolation method of Rinderknecht et al., J. Biol. Chem.259:6790 (1984), requires the binding of the interferon withconcanavalin A-sepharose in one step. Within additional embodiments ofthe invention, a fusion of the polypeptide of interest and an affinitytag (e.g., maltose-binding protein, an immnunoglobulin domain) may beconstructed to facilitate purification.

Zcyto13 polypeptides or fragments thereof may also be prepared throughchemical synthesis, as described below. Zcyto13 polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; PEGylated ornon-PEGylated; and may or may not include an initial methionine aminoacid residue.

8. Assays for Zcyto13, Its Analogs, and the Zcyto13 Receptor

As described above the disclosed polypeptides can be used to constructZcyto13 variants. A Zcyto13 variant may be functionally characterized byits ability to specifically bind with an anti-Zcyto13 antibody, or by abiological activity such as anti-viral activity, anti-proliferativeactivity, the ability to stimulate the expression of a gene known to beinduced by murine interferon-α (e.g., a xanthine dehydrogenase gene ofL929 fibroblastic cells, a 2′5′-oligoadenylate synthetase in quiescentBALB/c mouse 3T3 cells), or the ability to decrease the development ofspontaneous diabetes and the passive transfer of diabetes in NOD mousemodel of human IDDM. Illustrative activity assays are described below. Apolypeptide produced by a Zcyto13 variant gene is considered to be aZcyto13 agonist if the polypeptide exhibits a biological activity.

On the other hand, a Zcyto13 variant gene product that lacks biologicalactivity may be a Zcyto13 antagonist. These biologically-inactiveZcyto13 variants can be initially identified on the basis ofhybridization analysis, sequence identity determination, or by theability to specifically bind anti-Zcyto13 antibody. A Zcyto13 antagonistcan be further characterized by its ability to inhibit the biologicalresponse induced by Zcyto13 or by a Zcyto13 agonist. This inhibitoryeffect may result, for example, from the competitive or non-competitivebinding of the antagonist to the Zcyto13 receptor.

Zcyto13, its agonists and antagonists are valuable in both in vivo andin vitro uses. As an illustration, cytokines can be used as componentsof defined cell culture media, alone or in combination with othercytokines and hormones, to replace serum that is commonly used in cellculture. In particular, interferons have been shown to stimulate theproduction of other biologically active polypeptides, such asinterleukin-1, by cultured cells, which can be isolated from the culture(see, for example, Danis et al., Clin. Exp. Immunol. 80:435 (1990)).Interferons have also been shown to induce the expression of antigens bycultured cells (see, for example, Auth et al., Hepatology 18:546 (1993),Guadagni et al., Int. J. Biol. Markers 9:53 (1994), Girolomoni et al.,Eur. J. Immunol. 25:2163 (1995), and Maciejewski et al., Blood 85:3183(1995). This activity enhances the ability to identify new tumorassociated antigens in vitro. Moreover, the ability of interferons toaugment the level of expression of human tumor antigens indicates thatinterferons can be useful in an adjuvant setting for immunotherapy orimmunoscintigraphy using anti-tumor antigen antibodies (Guadagni et al.,Cancer Immunol. Immunother. 26:222 (1988); Guadagni et al., Int. J.Biol. Markers 9:53 (1994)).

Antagonists are also useful as research reagents for characterizingsites of interaction between Zcyto13 and its receptor. In a therapeuticsetting, pharmaceutical compositions comprising Zcyto13 antagonists canbe used to inhibit Zcyto13 activity.

One general class of Zcyto13 analogs are agonists or antagonists havingan amino acid sequence that is a mutation of the amino acid sequencesdisclosed herein. Another general class of Zcyto13 analogs is providedby anti-idiotype antibodies, and fragments thereof, as described below.Moreover, recombinant antibodies comprising anti-idiotype variabledomains can be used as analogs (see, for example, Monfardini et al.,Proc. Assoc. Am. Physicians 108:420 (1996)). Since the variable domainsof anti-idiotype Zcyto13 antibodies mimic Zcyto13, these domains canprovide either Zcyto13 agonist or antagonist activity. As anillustration, Lim and Langer, J. Interferon Res. 13:295 (1993), describeanti-idiotypic interferon-α antibodies that have the properties ofeither interferon-α agonists or antagonists.

A third approach to identifing Zcyto13 analogs is provided by the use ofcombinatorial libraries. Methods for constructing and screening phagedisplay and other combinatorial libraries are provided, for example, byKay et al., Phage Display of Peptides and Proteins (Academic Press1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et. al., U.S. Pat. No.5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.

One assay that can be used to measure Zcyto13 biological activity is aninterferon in vitro virus inhibition assay. For example, the anti-viralactivity of variant Zcyto13 polypeptides can be assessed by a cytopathiceffect reduction assay, in which mouse L929 cells are challenged withMengo virus or vesicular stomatitis virus (Stewart, The InterferonSystem (Springer 1979); Zawatzky et al., J. Gen. Virol. 63:325 (1982);Van Heuvel et al., J. Gen. Virol. 67:2215 (1986)). The anti-viralactivity of a variant Zcyto13 can also be measured with murinecytomegalovirus-infected NIH 3T3 fibroblasts (Gribaudo et al., Virology197:303 (1993)). Example 5 illustrates a method for testing anti-viralactivity using encephalomyocarditis virus and murine L929 cells, as wellas other methods for determining Zcyto13 activity.

Another approach to evaluating Zcyto13 activity is to use an assay thatmeasures the inhibition of the proliferation of cultured murine cells.For example, the anti-proliferative activity of a variant Zcyto13polypeptide can be determined with mouse B-16 melanoma cells(Fleischmann and Fleischmann, J. Biol. Regul. Homeost. Agents 2:173(1988)).

In another approach, Zcyto13 activity is measured by the ability of thetest polypeptide to stimulate the expression of a gene that is inducedby murine interferon-α. Illustrative genes include a xanthinedehydrogenase gene of L929 fibroblastic cells, and a 2′5′-oligoadenylatesynthetase in quiescent BALB/c mouse 3T3 cells (Falciani et al.,Biochem. J. 285:1001 (1992); Yan et al., Proc. Nat'l Acad. Sci USA86:2243 (1989)).

In yet another approach, the activity of a Zcyto13 variant is determinedusing an in vivo assay. For example, interferon-α administrationdecreases the development of spontaneous diabetes and the passivetransfer of diabetes in NOD mouse model of human IDDM (Sobel and Ahvazi,Diabetes 47:1867 (1998)).

As a receptor ligand, the activity of Zcyto13 can be measured by asilicon-based biosensor microphysiometer, which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent cellular responses. An exemplary deviceis the CYTOSENSOR Microphysiometer manufactured by Molecular DevicesCorp. (Sunnyvale, Calif.). A variety of cellular responses, such as cellproliferation, ion transport, energy production, inflammatory response,regulatory and receptor activation, and the like, can be measured bythis method (see, for example, McConnell et al., Science 257:1906(1992), Pitchford et al., Meth. Enzymol. 228:84 (1997), Arimilli et al.,J. Immunol. Meth. 212:49 (1998), and Van Liefde et al., Eur. J.Pharmacol. 346:87 (1998)). Moreover, the microphysiometer can be usedfor assaying adherent or non-adherent eukaryotic or prokaryotic cells.

Since energy metabolism is coupled with the use of cellular ATP, anyevent which alters cellular ATP levels, such as receptor activation andthe initiation of signal transduction, will cause a change in cellularacid section. An early event in interferon signal transduction isprotein phosphorylation, which requires ATP. By measuring extracellularacidification changes in cell media over time, therefore, themicrophysiometer directly measures cellular responses to variousstimuli, including Zcyto13, its agonists, or antagonists. Amicrophysiometer can be used to measure responses of a Zcyto13responsive eukaryotic cell, compared to a control eukaryotic cell thatdoes not respond to Zcyto13 polypeptide. Zcyto13 responsive eukaryoticcells comprise cells into which a receptor for Zcyto13 has beentransfected to create a cell that is responsive to Zcyto13, or cellsthat are naturally responsive to Zcyto13. Examples of murine cells thatrespond to murine interferon-α members include embryonic stem cells,B-16 melanoma cells, L₉₂₉ fibroblastic cells, and 3T3 cells (see, forexample, Fleischmann and Fleischmann, J. Biol. Regul. Homeost. Agents2:173 (1988); Yan et al., Proc. Nat'l Acad. Sci USA 86:2243 (1989);Falciani et al., Biochem. J. 285:1001 (1992); Whyatt et al., Mol. CellBiol. 13:7971 (1993)). Zcyto13 modulated cellular responses are measuredby a change (e.g., an increase or decrease in extracellularacidification) in the response of cells exposed to Zcyto13, comparedwith control cells that have not been exposed to Zcyto13.

Accordingly, a microphysiometer can be used to identify cells, tissues,or cell lines which respond to a Zcyto13 stimulated pathway, and whichexpress a functional Zcyto13 receptor. As an illustration, cells thatexpress a functional Zcyto13 receptor can be identified by (a) providingtest cells, (b) incubating a first portion of the test cells in theabsence of Zcyto13, (c) incubating a second portion of the test cells inthe presence of Zcyto13, and (d) detecting a change (e.g, an increase ordecrease in extracellular acidification rate, as measured by amicrophysiometer) in a cellular response of the second portion of thetest cells, as compared to the first portion of the test cells, whereinsuch a change in cellular response indicates that the test cells expressa functional Zcyto13 receptor. An additional negative control may beincluded in which a portion of the test cells is incubated with Zcyto13and an anti-Zcyto13 antibody to inhibit the binding of Zcyto13 with itscognate receptor.

The microphysiometer also provides one means to identify Zcyto13agonists. For example, agonists of Zcyto13 can be identified by amethod, comprising the steps of (a) providing cells responsive toZcyto13, (b) incubating a first portion of the cells in the absence of atest compound, (c) incubating a second portion of the cells in thepresence of a test compound, and (d) detecting a change, for example, anincrease or diminution, in a cellular response of the second portion ofthe cells as compared to the first portion of the cells, wherein such achange in cellular response indicates that the test compound is aZcyto13 agonist. An illustrative change in cellular response is ameasurable change in extracellular acidification rate, as measured by amicrophysiometer. Moreover, incubating a third portion of the cells inthe presence of Zcyto13 and in the absence of a test compound can beused as a positive control for the Zcyto13 responsive cells, and as acontrol to compare the agonist activity of a test compound with that ofZcyto13. An additional control may be included in which a portion of thecells is incubated with a test compound (or Zcytol13) and ananti-Zcyto13 antibody to inhibit the binding of the test compound (orZcytol13) with the Zcyto13 receptor.

The microphysiometer also provides a means to identify Zcyto13antagonists. For example, Zcyto13 antagonists can be identified by amethod, comprising the steps of (a) providing cells responsive toZcyto13, (b) incubating a first portion of the cells in the presence ofZcyto13 and in the absence of a test compound, (c) incubating a secondportion of the cells in the presence of both Zcyto13 and the testcompound, and (d) comparing the cellular responses of the first andsecond cell portions, wherein a decreased response by the secondportion, compared with the response of the first portion, indicates thatthe test compound is a Zcyto13 antagonist. An illustrative change incellular response is a measurable change extracellular acidificationrate, as measured by a microphysiometer.

Zcyto13, its analogs, and anti-iodiotype Zcyto13 antibodies can be usedto identify and to isolate Zcyto13 receptors. For example, proteins andpeptides of the present invention can be immobilized on a column andused to bind receptor proteins from membrane preparations that are runover the column (Hermanson et al. (eds.), Immobilized Affinity LigandTechniques, pages 195-202 (Academic Press 1992)). Radiolabeled oraffinity labeled Zcyto13 polypeptides can also be used to identify or tolocalize Zcyto13 receptors in a biological sample (see, for example,Deutscher (ed.), Methods in Enzymol., vol. 182, pages 721-37 (AcademicPress 1990); Brunner et al., Ann. Rev. Biochem. 62:483 (1993); Fedan etal., Biochem. Pharmacol. 33:1167 (1984)). Also see, Varthakavi andMinocha, J. Gen. Virol. 77:1875 (1996), who describe the use ofanti-idiotype antibodies for receptor identification.

9. Production of Antibodies to Zcyto13 Proteins

Antibodies to Zcyto13 can be obtained, for example, using the product ofa Zcyto13 expression vector or Zcyto13 isolated from a natural source asan antigen. Particularly useful anti-Zcyto13 antibodies “bindspecifically” with Zcyto13. Antibodies are considered to be specificallybinding if the antibodies exhibit at least one of the following twoproperties: (1) antibodies bind to Zcyto13 with a threshold level ofbinding activity, and (2) antibodies do not significantly cross-reactwith polypeptides related to Zcyto13.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a Zcyto13 polypeptide, peptide or epitope with a bindingaffinity (K_(a)) of 10⁶M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). With regard to the secondcharacteristic, antibodies do not significantly cross-react with relatedpolypeptide molecules, for example, if they detect Zcyto13, but notknown polypeptides using a standard Western blot analysis. Examples ofknown related polypeptides are orthologs and proteins from the samespecies that are members of a protein family. For example,specifically-binding anti-Zcyto13 antibodies bind with Zcyto13, but notwith polypeptides such as other interferon-α polypeptides, interferon-β,interferon-γ, interferon-δ, interferon-ω, or interferon-τ. Suitableantibodies include antibodies that bind with Zcyto13 in regions having alow sequence similarity with other interferons.

Anti-Zcyto13 antibodies can be produced using antigenic Zcyto13epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, or between 15 to about 30 amino acids contained withinSEQ ID NO:2. However, peptides or polypeptides comprising a largerportion of an amino acid sequence of the invention, containing from 30to 50 amino acids, or any length up to and including the entire aminoacid sequence of a polypeptide of the invention, also are useful forinducing antibodies that bind with Zcyto13. It is desirable that theamino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (ie., the sequenceincludes relatively hydrophilic residues, while hydrophobic residues arepreferably avoided). Moreover, amino acid sequences containing prolineresidues may be also be desirable for antibody production.

As an illustration, potential antigenic sites in Zcyto13 were identifiedusing the Jameson-Wolf method, Jameson and Wolf, CABIOS 4:181, (1988),as implemented by the PROTEAN program (version 3.14) of LASERGENE(DNASTAR; Madison, Wis.). Default parameters were used in this analysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Garnier-Robson, Gamnier et al., J. Mol. Biol.120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; αregion threshold=103; β region threshold=105; Garnier-Robson parameters:α and β decision constants=0). In the sixth subroutine, flexibilityparameters and hydropathy/solvent accessibility factors were combined todetermine a surface contour value, designated as the “antigenic index.”Finally, a peak broadening function was applied to the antigenic index,which broadens major surface peaks by adding 20, 40, 60, or 80% of therespective peak value to account for additional free energy derived fromthe mobility of surface regions relative to interior regions. Thiscalculation was not applied, however, to any major peak that resides ina helical region, since helical regions tend to be less flexible.

The results of this analysis indicated that the following amino acidsequences of SEQ ID NO:2 would provide suitable antigenic peptides:amino acids 51 to 61 (“antigenic peptide 1”), amino acids 68 to 73(“antigenic peptide 2”), amino acids 90 to 96 (“antigenic peptide 3”),amino acids 97 to 136 (“antigenic peptide 4”), amino acids 104 to 110(“antigenic peptide 5”), amino acids 120 to 136 (“antigenic peptide 6”),amino acids 156 to 161 (“antigenic peptide 7”), amino acids 179 to 185(“antigenic peptide 8”), and amino acids 190 to 199 (“antigenic peptide9”). The present invention contemplates the use of any one of antigenicpeptides 1 to 9 to generate antibodies to Zcyto13. The present inventionalso contemplates polypeptides comprising at least one of antigenicpeptides 1 to 9.

Polyclonal antibodies to recombinant Zcyto13 protein or to Zcyto13isolated from natural sources can be prepared using methods well-knownto those of skill in the art. See, for example, Green et al.,“Production of Polyclonal Antisera,” in Immunochemical Protocols(Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995). The immunogenicity of a Zcyto13 polypeptide canbe increased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of Zcyto13 or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. The polypeptide immunogenmay be a full-length molecule or a portion thereo. If the polypeptideportion is “hapten-like,” such portion may be advantageously joined orlinked to a macromolecular carrier (such as keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, orsheep, an anti-Zcyto13 antibody of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingdiagnostically and therapeutically useful antibodies in baboons may befound, for example, in Goldenberg et al., international patentpublication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310(1990).

Alternatively, monoclonal anti-Zcyto13 antibodies can be generated.Rodent monoclonal antibodies to specific antigens may be obtained bymethods known to those skilled in the art (see, for example, Kohler etal., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a Zcyto13 gene product, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

In addition, an anti-Zcyto13 antibody of the present invention may bederived from a human monoclonal antibody. Human monoclonal antibodiesare obtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-Zcyto13 antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem.J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise V_(H) and V_(L) chains, which areconnected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains which areconnected by an oligonucleotide. The structural gene is inserted into anexpression vector which is subsequently introduced into a host cell,such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are described, for example, by Whitlow etal., Methods: A Companion to Methods in Enzymology 2:97 (1991) (alsosee, Bird et al., Science 242:423 (1988), Ladner et al., U.S. Pat. No.4,946,778, Pack et al., Bio/Technology 11:1271 (1993), and Sandhu,supra).

As an illustration, an scFV can be obtained by exposing lymphocytes toZcyto13 polypeptide in vitro, and selecting antibody display librariesin phage or similar vectors (for instance, through use of immobilized orlabeled Zcyto13 protein or peptide). Genes encoding polypeptides havingpotential Zcyto13 polypeptide binding domains can be obtained byscreening random peptide libraries displayed on phage (phage display) oron bacteria, such as E coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such randompeptide display libraries are known in the art (Ladner et al., U.S. Pat.No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al.,U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kayet al., Phage Display of Peptides and Proteins (Academic Press, Inc.1996)) and random peptide display libraries and kits for screening suchlibraries are available commercially, for instance from CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego,Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). Random peptide display librariescan be screened using the Zcyto13 sequences disclosed herein to identifyproteins which bind to Zcyto13.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-Zcyto13 antibody may be derived from a“humanized” monoclonal antibody. Humanized monoclonal antibodies areproduced by transferring mouse complementary determining regions fromheavy and light variable chains of the mouse immunoglobulin into a humanvariable domain. Typical residues of human antibodies are thensubstituted in the framework regions of the murine counterparts. The useof antibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833 (1989). Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad Sci. USA 89:4285(1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J.Immun. 150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols(Humana Press, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,”in Protein Engineering: Principles and Practice, Cleland et al (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-Zcyto13 antibodies or antibody fragments, usingstandard techniques. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, seeColigan at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotypeantibodies can be prepared using anti-Zcyto13 antibodies or antibodyfragments as immunogens with the techniques, described above. As anotheralternative, humanized anti-idiotype antibodies or subhuman primateanti-idiotype antibodies can be prepared using the above-describedtechniques. Methods for producing anti-idiotype antibodies aredescribed, for example, by Irie, U.S. Pat. No. 5,208,146, Greene, et.al., U.S. Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol.77:1875 (1996).

10. Use of Zcyto13 Nucleotide Sequences to Detect Zcyto13 GeneExpression and to Examine Zcyto13 Gene Structure

Nucleic acid molecules can be used to detect the expression of a Zcyto13gene in a biological sample. Suitable probe molecules includedouble-stranded nucleic acid molecules comprising the nucleotidesequence of SEQ ID NO:1, or a portion thereof, as well assingle-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NO:1, or a portion thereof. Probemolecules may be DNA, RNA, oligonucleotides, and the like. As usedherein, the term “portion” refers to at least eight nucleotides to atleast 20 or more nucleotides. Certain probes bind with regions of theZcyto13 gene that have a low sequence similarity to comparable regionsin other interferons.

In a basic assay, a single-stranded probe molecule is incubated withRNA, isolated from a biological sample, under conditions of temperatureand ionic strength that promote base pairing between the probe andtarget Zcyto13 RNA species. After separating unbound probe fromhybridized molecules, the amount of hybrids is detected.

Well-established hybridization methods of RNA detection include northernanalysis and dot/slot blot hybridization (see, for example, Ausubel(1995) at pages 4-1 to 4-27, and Wu et al. (eds.), “Analysis of GeneExpression at the RNA Level,” in Methods in Gene Biotechnology, pages225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be detectablylabeled with radioisotopes such as ³²P or ³⁵S. Alternatively, Zcyto13RNA can be detected with a nonradioactive hybridization method (see, forexample, Isaac (ed.), Protocols for Nucleic Acid Analysis byNonradioactive Probes (Humana Press, Inc. 1993)). Typically,nonradioactive detection is achieved by enzymatic conversion ofchromogenic or chemiluminescent substrates. Illustrative nonradioactivemoieties include biotin, fluorescein, and digoxigenin.

Zcyto13 oligonucleotide probes are also usefwll for in vivo diagnosis.As an illustration, ¹⁸F-labeled oligonucleotides can be administered toa subject and visualized by positron emission tomography (Tavitian etal., Nature Medicine 4:467 (1998)).

Numerous diagnostic procedures take advantage of the polymerase chainreaction (PCR) to increase sensitivity of detection methods. Standardtechniques for performing PCR are well-known (see, generally, Mathew(ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),White (ed.), PCR Protocols: Current Methods and Applications (HumanaPress, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (HumanaPress, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (HumanaPress, Inc. 1998)).

PCR primers can be designed to amplify a portion of the Zcyto13 genethat has a low sequence similarity to a comparable region in otherinterferons.

One variation of PCR for diagnostic assays is reverse transcriptase-PCR(RT-PCR). In the RT-PCR technique, RNA is isolated from a biologicalsample, reverse transcribed to cDNA, and the cDNA is incubated withZcyto13 primers (see, for example, Wu et al. (eds.), “Rapid Isolation ofSpecific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, pages15-28 (CRC Press, Inc. 1997)). PCR is then performed and the productsare analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, forexample, the gunadinium-thiocyanate cell lysis procedure describedabove. Alternatively, a solid-phase technique can be used to isolatemRNA from a cell lysate. A reverse transcription reaction can be primedwith the isolated RNA using random oligonucleotides, short homopolymersof dT, or Zcyto13 anti-sense oligomers. Oligo-dT primers offer theadvantage that various mRNA nucleotide sequences are amplified that canprovide control target sequences. Zcyto13 sequences are amplified by thepolymerase chain reaction using two flanking oligonucleotide primersthat are typically 20 bases in length.

PCR amplification products can be detected using a variety ofapproaches. For example, PCR products can be fractionated by gelelectrophoresis, and visualized by ethidium bromide staining.Alternatively, fractionated PCR products can be transferred to amembrane, hybridized with a detectably-labeled Zcyto13 probe, andexamined by autoradiography. Additional alternative approaches includethe use of digoxigenin-labeled deoxyribonucleic acid triphosphates toprovide chemiluminescence detection, and the C-TRAK calorimetric assay.

Another approach for detection of Zcyto13 expression is cycling probetechnology (CPT), in which a single-stranded DNA target binds with anexcess of DNA-RNA-DNA chimeric probe to form a complex, the RNA portionis cleaved with RNAase H, and the presence of cleaved chimeric probe isdetected (see, for example, Beggs et al., J. Clin. Microbiol. 34:2985(1996), Bekkaoui et al., Biotechniques 20:240 (1996)). Alternativemethods for detection of Zcyto13 sequences can utilize approaches suchas nucleic acid sequence-based amplification (NASBA), cooperativeamplification of templates by cross-hybridization (CATCH), and theligase chain reaction (LCR) (see, for example, Marshall et al., U.S.Pat. No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 60:161 (1996),Ehricht et al., Eur. J. Biochem. 243:358 (1997), and Chadwick et al., J.Virol. Methods 70:59 (1998)). Other standard methods are known to thoseof skill in the art.

Zcyto13 probes and primers can also be used to detect and to localizeZcyto13 gene expression in tissue samples. Methods for such in situhybridization are well-known to those of skill in the art (see, forexample, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc.1994), Wu et al. (eds.), “Analysis of Cellular DNA or Abundance of mRNAby Radioactive In Situ Hybridization (RISH),” in Methods in GeneBiotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.(eds.), “Localization of DNA or Abundance of mRNA by Fluorescence InSitu Hybridization (RISH),” in Methods in Gene Biotechnology, pages279-289 (CRC Press, Inc. 1997)). Various additional diagnosticapproaches are well-known to those of skill in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (HumanaPress, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases(Humana Press, Inc., 1996)).

Nucleic acid molecules comprising Zcyto13 nucleotide sequences can alsobe used to determine whether a subject's chromosomes contain a mutationin the Zcyto13 gene. Detectable chromosomal aberrations at the Zcyto13gene locus include, but are not limited to, aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. Of particular interest are genetic alterations thatinactivate the Zcyto13 gene.

Aberrations associated with the Zcyto13 locus can be detected usingnucleic acid molecules of the present invention by employing moleculargenetic techniques, such as restriction fragment length polymorphismanalysis, short tandem repeat analysis employing PCR techniques,amplification-refractory mutation system analysis, single-strandconformation polymorphism detection, RNase cleavage methods, denaturinggradient gel electrophoresis, fluorescence-assisted mismatch analysis,and other genetic analysis techniques known in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis,Molecular Diagnostics (Human Press, Inc. 1996), Elles (ed.) MolecularDiagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren(ed.), Laboratory Protocols for Mutation Detection (Oxford UniversityPress 1996), Birren et al. (eds.), Genome Analysis, Vol. 2: DetectingGenes (Cold Spring Harbor Laboratory Press 1998), Dracopoli et al.(eds.), Current Protocols in Human Genetics (John Wiley & Sons 1998),and Richards and Ward, “Molecular Diagnostic Testing,” in Principles ofMolecular Medicine, pages 83-88 (Humana Press, Inc. 1998)).

The protein truncation test is also useful for detecting theinactivation of a gene in which translation-terminating mutationsproduce only portions of the encoded protein (see, for example,Stoppa-Lyonnet et al., Blood 91:3920 (1998)). According to thisapproach, RNA is isolated from a biological sample, and used tosynthesize cDNA. PCR is then used to amplify the Zcyto13 target sequenceand to introduce an RNA polymerase promoter, a translation initiationsequence, and an in-frame ATG triplet. PCR products are transcribedusing an RNA polymerase, and the transcripts are translated in vitrowith a T7-coupled reticulocyte lysate system. The translation productsare then fractionated by SDS-PAGE to determine the lengths of thetranslation products. The protein truncation test is described, forexample, by Dracopoli et al. (eds.), Current Protocols in HumanGenetics, pages 9.11.1-9.11.18 (John Wiley & Sons 1998).

The present invention also contemplates kits for performing a diagnosticassay for Zcyto13 gene expression or to detect mutations in the Zcyto13gene. Such kits comprise nucleic acid probes, such as double-strandednucleic acid molecules comprising the nucleotide sequence of SEQ IDNO:1, or a portion thereof, as well as single-stranded nucleic acidmolecules having the complement of the nucleotide sequence of SEQ IDNO:1, or a portion thereof. Probe molecules may be DNA, RNA,oligonucleotides, and the like. Kits may comprise nucleic acid primersfor performing PCR.

Such a kit can contain all the necessary elements to perform a nucleicacid diagnostic assay described above. A kit will comprise at least onecontainer comprising a Zcyto13 probe or primer. The kit may alsocomprise a second container comprising one or more reagents capable ofindicating the presence of Zcyto13 sequences. Examples of such indicatorreagents include detectable labels such as radioactive labels,fluorochromes, chemiluminescent agents, and the like. A kit may alsocomprise a means for conveying to the user that the Zcyto13 probes andprimers are used to detect Zcyto13 gene expression. For example, writteninstructions may state that the enclosed nucleic acid molecules can beused to detect either a nucleic acid molecule that encodes Zcyto13, or anucleic acid molecule having a nucleotide sequence that is complementaryto a Zcyto13-encoding nucleotide sequence. The written material can beapplied directly to a container, or the written material can be providedin the form of a packaging insert.

11. Use of Anti-Zcyto13 Antibodies to Detect Zcyto13 Protein

The present invention contemplates the use of anti-Zcyto13 antibodies toscreen biological samples in vitro for the presence of Zcyto13. In onetype of in vitro assay, anti-Zcyto13 antibodies are used in liquidphase. For example, the presence of Zcyto13 in a biological sample canbe tested by mixing the biological sample with a trace amount of labeledZcyto13 and an anti-Zcyto13 antibody under conditions that promotebinding between Zcyto13 and its antibody. Complexes of Zcyto13 andanti-Zcyto13 in the sample can be separated from the reaction mixture bycontacting the complex with an immobilized protein which binds with theantibody, such as an Fc antibody or Staphylococcus protein A. Theconcentration of Zcyto13 in the biological sample will be inverselyproportional to the amount of labeled Zcyto13 bound to the antibody anddirectly related to the amount of free labeled Zcyto13.

Alternatively, in vitro assays can be performed in which anti- Zcyto13antibody is bound to a solid-phase carrier. For example, antibody can beattached to a polymer, such as aminodextran, in order to link theantibody to an insoluble support such as a polymer-coated bead, a plateor a tube. Other suitable in vitro assays will be readily apparent tothose of skill in the art.

In another approach, anti-Zcyto13 antibodies can be used to detectZcyto13 in tissue sections prepared from a biopsy specimen. Suchimmunochemical detection can be used to determine the relative abundanceof Zcyto13 and to determine the distribution of Zcyto13 in the examinedtissue. General immunochemistry techniques are well established (see,for example, Ponder, “Cell Marking Techniques and Their Application,” inMammalian Development: A Practical Approach, Monk (ed.), pages 115-38(IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages14.6.1 to 14.6.13 (Wiley Interscience 1990), and Manson (ed.), MethodsIn Molecular Biology, Vol. 10: Immunochemical Protocols (The HumanaPress, Inc. 1992)).

Immunochemical detection can be performed by contacting a biologicalsample with an anti-Zcyto13 antibody, and then contacting the biologicalsample with a detectably labeled molecule which binds to the antibody.For example, the detectably labeled molecule can comprise an antibodymoiety that binds to anti-Zcyto13 antibody. Alternatively, theanti-Zcyto13 antibody can be conjugated with avidin/streptavidin (orbiotin) and the detectably labeled molecule can comprise biotin (oravidin/streptavidin). Numerous variations of this basic technique arewell-known to those of skill in the art.

Alternatively, an anti-Zcyto13 antibody can be conjugated with adetectable label to form an anti-Zcyto13 immunoconjugate. Suitabledetectable labels include, for example, a radioisotope, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescent labelor colloidal gold. Methods of making and detecting suchdetectably-labeled immunoconjugates are well-known to those of ordinaryskill in the art, and are described in more detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-Zcyto13 immunoconjugates can also be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody is determinedby exposing the immunoconjugate to light of the proper wavelength anddetecting the resultant fluorescence. Fluorescent labeling compoundsinclude fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Alternatively, anti-Zcyto13 immunoconjugates can be detectably labeledby coupling an antibody component to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged immunoconjugate is determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-Zcyto13immunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, anti-Zcyto13 immunoconjugates can be detectably labeledby linking an anti-Zcyto13 antibody component to an enzyme. When theanti-Zcyto13-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-Zcyto13 antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al., Clin. Chim. Acta 70:1 (1976), Schurs etal., Clin. Chim. Acta 81:1 (1977), Shih et al., Int'l J. Cancer 46:1101(1990), Stein et al., Cancer Res. 50:1330 (1990), and Coligan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-Zcyto13 antibodies that have beenconjugated with avidin, streptavidin, and biotin (see, for example,Wilchek et al (eds.), “Avidin-Biotin Technology,” Methods In Enzymology,Vol. 184 (Academic Press 1990), and Bayer et al., “ImmunochemicalApplications of Avidin-Biotin Technology,” in Methods In MolecularBiology, Vol. 10, Manson (ed.), pages 149-162 (The Humana Press, Inc.1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

In a related approach, biotin- or FITC-labeled Zcyto13 can be used toidentify cells that bind Zcyto13. Such can binding can be detected, forexample, using flow cytometry.

The present invention also contemplates kits for performing animmunological diagnostic assay for Zcyto13 gene expression. Such kitscomprise at least one container comprising an anti-Zcyto13 antibody, orantibody fragment. A kit may also comprise a second container comprisingone or more reagents capable of indicating the presence of Zcyto13antibody or antibody fragments. Examples of such indicator reagentsinclude detectable labels such as a radioactive label, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescentlabel, colloidal gold, and the like. A kit may also comprise a means forconveying to the user that Zcyto13 antibodies or antibody fragments areused to detect Zcyto13 protein. For example, written instructions maystate that the enclosed antibody or antibody fragment can be used todetect Zcyto13. The written material can be applied directly to acontainer, or the written material can be provided in the form of apackaging insert.

12. Therapeutic Uses of Polypeptides Having Zcyto13 Activity

Interferons are known to be potent cytokines that possess antiviral,immunomodulating, and anti-proliferative activities. Therefore, thepresent invention includes the use of proteins, polypeptides, andpeptides having Zcyto13 activity (such as Zcyto13 polypeptides, Zcyto13analogs, and Zcyto13 fusion proteins) to provide antiviral,immunomodulatory, or anti-proliferative activity. The present inventioncontemplates the use of these molecules for either veterinary or forhuman therapeutic uses.

Both recombinant interferons and interferons isolated from naturalsources have been approved in the United States for treatment ofautoimmune diseases, renal cell carcinoma, basal cell carcinoma,malignant melanoma, condylomata acuminata, chronic hepatitis B,hepatitis C, chronic hepatitis D, chronic non-A, non-B/C hepatitis,bladder carcinoma, multiple myeloma, chronic myelogenous leukemia,non-Hodgkin's lymphoma, cervical carcinoma, laryngeal papilloma,fungoides mycosis, Kaposi's sarcoma in patients infected with humanimmunodeficiency virus, hairy cell leukemia, and multiple sclerosis. Inaddition, Zcyto13 may be used to treat forms of arteriosclerosis, suchas atherosclerosis, by inhibiting cell proliferation, or to treatretinopathy. Accordingly, the present invention contemplates the use ofproteins, polypeptides, and peptides having Zcyto13 activity to treatsuch conditions.

In addition, interferons are known to augment the level of expression ofhuman tumor antigens, as discussed above. Thus, the present inventionincludes the use of proteins, polypeptides and peptides having Zcyto13activity as an adjuvant for immunotherapy or immunoscintigraphy usinganti-tumor antigen antibodies.

Generally, the dosage of administered Zcyto13 (or Zcyto13 analog orfusion protein) will vary depending upon such factors as the subject'sage, weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of Zcyto13, which is in the range of from about 1 pg/kg to 10mg/kg (amount of agent/body weight of subject), although a lower orhigher dosage also may be administered as circumstances dictate.

Administration of a molecule having Zcyto13 activity to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising Zcyto13can be prepared and inhaled with the aid of dry-powder dispersers,liquid aerosol generators, or nebulizers (e.g., Pettit and Gombotz,TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235(1999)). This approach is illustrated by the AERX diabetes managementsystem, which is a hand-held electronic inhaler that deliversaerosolized insulin into the lungs. Studies have shown that proteins aslarge as 48,000 kDa have been delivered across skin at therapeuticconcentrations with the aid of low-frequency ultrasound, whichillustrates the feasibility of trascutaneous administration (Mitragotriet al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer a molecule havingZcyto13 activity (Potts et al., Pharm. Biotechnol. 10:213 (1997)).

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having Zcyto13 activity can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby thetherapeutic proteins are combined in a mixture with a pharmaceuticallyacceptable carrier. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient subject. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable carrier. Other suitable carriers arewell-known to those in the art. See, for example, Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995).

For purposes of therapy, molecules having Zcyto13 activity and apharmaceutically acceptable carrier are administered to a subject in atherapeutically effective amount. A combination of a protein,polypeptide, or peptide having Zcyto13 activity and a pharmaceuticallyacceptable carrier is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient subject.In the present context, an agent is physiologically significant if itspresence results in the inhibition of the growth of tumor cells or inthe inhibition of a viral infection. An inhibition of tumor growth maybe indicated, for example, by a decrease in the number of tumor cells,decreased metastasis, a decrease in the size of a solid tumor, orincreased necrosis of a tumor. Indicators of viral infection inhibitioninclude decreased viral titer, a decrease in detectable viral antigen,or an increase in anti-viral antibody titer.

A pharmaceutical composition comprising Zcyto13 (or Zcyto13 analog orfusion protein) can be fumished in liquid form, in an aerosol, or insolid form. Liquid forms, are illustrated by injectable solutions andoral suspensions. Exemplary solid forms include capsules, tablets, andcontrolled-release forms. The latter form is illustrated by miniosmoticpumps and implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997);Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems, Ranadeand Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,“Protein Delivery with Infusion Pumps,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 93-117 (Plenum Press 1997)). For example, Cleland andJones, Pharm. Res. 13:1464 (1996), describe a method for producinginterferon-γ encapsulated in polylactic-coglycolic microspheres.

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:1.33 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull.20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Polypeptides having Zcyto13 activity can be encapsulated withinliposomes using standard techniques of protein microencapsulation (see,for example, Anderson et al., Infect. Immun. 31:1099 (1981), Anderson etal., Cancer Res. 50:1853 (1990), and Cohen et al., Biochim. Biophys.Acta 1063:95 (1991), Alving et al. “Preparation and Use of Liposomes inImmunological Studies,” in Liposome Technology, 2nd Edition, Vol. III,Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol. 149:124 (1987)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al,Biochim. Biophys. Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.).pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modified polypeptideshaving Zcyto13 activity and Zcyto13 antagonists, in which a polypeptideis linked with a polymer, as discussed above.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a molecule having Zcyto13 activityor a Zcyto13 antagonist. Therapeutic polypeptides can be provided in theform of an injectable solution for single or multiple doses, or as asterile powder that will be reconstituted before injection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of a therapeuticpolypeptide. Such a kit may further comprise written information onindications and usage of the pharmaceutical composition. Moreover, suchinformation may include a statement that the Zcyto13 composition iscontraindicated in subjects with known hypersensitivity to Zcyto13.

13. Therapeutic Uses of Zcyto13 Nucleotide Sequences

Immunomodulator genes can be introduced into a subject to enhanceimmunological responses. As an illustration “immunomodulator genetherapy” has been examined in model systems using vectors that expressIL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, interferon-γ, tumornecrosis factor-α, or granulocyte-macrophage colony-stimulating factor(see, for example, Cao et al., J. Gastroenterol. Hepatol. 11:1053(1996), Tahara et al., Ann. N.Y Acad Sci. 795:275 (1996), Rakhmilevichet al., Hum. Gene Ther. 8:1303 (1997), and Cao et al., Transplantation65:325 (1998)). The present invention includes the use of Zcyto13nucleotide sequences to augment an immunological response to a tumor orviral infection in a subject. In addition, a therapeutic expressionvector can be provided that inhibits Zcyto13 gene expression, such as ananti-sense molecule, a ribozyme, or an external guide sequence molecule.

There are numerous approaches to introduce a Zcyto13 gene to a subject,including the use of recombinant host cells that express Zcyto13,delivery of naked nucleic acid encoding Zcyto13, use of a cationic lipidcarrier with a nucleic acid molecule that encodes Zcyto13, and the useof viruses that express Zcyto13, such as recombinant retroviruses,recombinant adeno-associated viruses, recombinant adenoviruses, andrecombinant Herpes simplex viruses [HSV] (see, for example, Mulligan,Science 260:926 (1993), Rosenberg et al., Science 242:1575 (1988),LaSalle et al., Science 259:988 (1993), Wolff et al., Science 247:1465(1990), Breakfield and Deluca, The New Biologist 3:203 (1991)). In an exvivo approach, for example, cells are isolated from a subject,transfected with a vector that expresses a Zcyto13 gene, and thentransplanted into the subject.

In order to effect expression of a Zcyto13 gene, an expression vector isconstructed in which a nucleotide sequence encoding a Zcyto13 gene isoperably linked to a core promoter, and optionally a regulatory element,to control gene transcription. The general requirements of an expressionvector are described above.

Alternatively, a Zcyto13 gene can be delivered using recombinant viralvectors, including for example, adenoviral vectors (e.g., Kass-Eisler etal., Proc. Nat'l Acad. Sci. USA 90:11498 (1993), Kolls et al., Proc.Nat'l Acad. Sci. USA 91:215 (1994), Li et al., Hum. Gene Ther. 4:403(1993), Vincent et al., Nat. Genet. 5:130 (1993), and Zabner et al.,Cell 75:207 (1993)), adenovirus-associated viral vectors (Flotte et al.,Proc. Nat'l Acad. Sci. USA 90:10613 (1993)), alphaviruses such asSemliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat. Nos.4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors(Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus vectors(Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali andPaoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)), pox viruses, suchas canary pox virus or vaccinia virus (Fisher-Hoch et al., Proc. Nat'lAcad. Sci. USA 86:317 (1989), and Flexner et al., Ann. N.Y Acad. Sci.569:86 (1989)), and retroviruses (e.g., Baba et al., J. Neurosurg 79:729(1993), Ram et al., Cancer Res. 53:83 (1993), Takamiya et al., J.Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993),Vile and Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S.Pat. No. 5,399,346). Within various embodiments, either the viral vectoritself, or a viral particle which contains the viral vector may beutilized in the methods and compositions described below.

As an illustration of one system, adenovirus, a double-stranded DNAvirus, is a well-characterized gene transfer vector for delivery of aheterologous nucleic acid molecule (for a review, see Becker et al.,Meth. Cell Biol. 43:161 (1994); Douglas and Curiel, Science & Medicine4:44 (1997)). The adenovirus system offers several advantages including:(i) the ability to accommodate relatively large DNA inserts, (ii) theability to be grown to high-titer, (iii) the ability to infect a broadrange of mammalian cell types, and (iv) the ability to be used with manydifferent promoters including ubiquitous, tissue specific, andregulatable promoters. In addition, adenoviruses can be administered byintravenous injection, because the viruses are stable in thebloodstream.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene is deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell. When intravenously administered to intact animals,adenovirus primarily targets the liver. Although an adenoviral deliverysystem with an E1 gene deletion cannot replicate in the host cells, thehost's tissue will express and process an encoded heterologous protein.Host cells will also secrete the heterologous protein if thecorresponding gene includes a secretory signal sequence. Secretedproteins will enter the circulation from tissue that expresses theheterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genescan be used to reduce or eliminate immune responses to the vector. Suchadenoviruses are E1-deleted, and in addition, contain deletions of E2Aor E4 (Lusky et al., J. Virol. 72:2022 (1998); Raper et al., Human GeneTherapy 9:671 (1998)). The deletion of E2b has also been reported toreduce immune responses (Amalfitano et al., J. Virol. 72:926 (1998)). Bydeleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses, where all viral genes are deleted, are particularlyadvantageous for insertion of large inserts of heterologous DNA (for areview, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing atherapeutic gene can be obtained from infected mammalian cells usingstandard methods. For example, recombinant HSV can be prepared in Verocells, as described by Brandt et al., J. Gen. Virol. 72:2043 (1991),Herold et al., J. Gen. Virol. 75:1211 (1994), Visalli and Brandt,Virology 185:419 (1991), Grau et al., Invest. Ophthalmol. Vis. Sci.30:2474 (1989), Brandt et al., J. Virol. Meth. 36:209 (1992), and byBrown and MacLean (eds.), HSV Virus Protocols (Humana Press 1997).

Alternatively, an expression vector comprising a Zcyto13 gene can beintroduced into a subject's cells by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey et al., Proc. Nat'lAcad. Sci. USA 85:8027 (1988)). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Liposomes can be used to direct transfection to particularcell types, which is particularly advantageous in a tissue with cellularheterogeneity, such as the pancreas, liver, kidney, and brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides (e.g., hormones or neurotransmitters),proteins such as antibodies, or non-peptide molecules can be coupled toliposomes chemically.

Electroporation is another alternative mode of administration. Forexample, Aihara and Miyazaki, Nature Biotechnology 16:867 (1998), havedemonstrated the use of in vivo electropotation for gene transfer intomuscle.

In an alternative approach to gene therapy, a therapeutic gene mayencode a Zcyto13 anti-sense RNA that inhibits the expression of Zcyto13.Suitable sequences for anti-sense molecules can be derived from thenucleotide sequences of Zcyto13 disclosed herein.

Alternatively, an expression vector can be constructed in which aregulatory element is operably linked to a nucleotide sequence thatencodes a ribozyme. Ribozymes can be designed to express endonucleaseactivity that is directed to a certain target sequence in a mRNAmolecule (see, for example, Draper and Macejak, U.S. Pat. No. 5,496,698,McSwiggen, U.S. Pat. No. 5,525,468, Chowrira and McSwiggen, U.S. Pat.No. 5,631,359, and Robertson and Goldberg, U.S. Pat. No. 5,225,337). Inthe context of the present invention, ribozymes include nucleotidesequences that bind with Zcyto13 mRNA.

In another approach, expression vectors can be constructed in which aregulatory element directs the production of RNA transcripts capable ofpromoting RNase P-mediated cleavage of mRNA molecules that encode aZcyto13 gene. According to this approach, an external guide sequence canbe constructed for directing the endogenous ribozyme, RNase P, to aparticular species of intracellular mRNA, which is subsequently cleavedby the cellular ribozyme (see, for example, Altman et al., U.S. Pat. No.5,168,053, Yuan et al., Science 263:1269 (1994), Pace et al.,international publication No. WO 96/18733, George et al., internationalpublication No. WO 96/21731, and Werner et al., internationalpublication No. WO 97/33991). Preferably, the external guide sequencecomprises a ten to fifteen nucleotide sequence complementary to Zcyto13mRNA, and a 3′-NCCA nucleotide sequence, wherein N is preferably apurine. The external guide sequence transcripts bind to the targetedmRNA species by the formation of base pairs between the mRNA and thecomplementary external guide sequences, thus promoting cleavage of mRNAby RNase P at the nucleotide located at the 5′-side of the base-pairedregion.

In general, the dosage of a composition comprising a therapeutic vectorhaving a Zcyto13 nucleotide acid sequence, such as a recombinant virus,will vary depending upon such factors as the subject's age, weight,height, sex, general medical condition and previous medical history.Suitable routes of administration of therapeutic vectors includeintravenous injection, intraarterial injection, intraperitonealinjection, intramuscular injection, intratumoral injection, andinjection into a cavity that contains a tumor. As an illustration,Horton et al., Proc. Nat'l Acad. Sci. USA 96:1553 (1999), demonstratedthat intramuscular injection of plasmid DNA encoding interferon-αproduces potent antitumor effects on primary and metastatic tumors in amurine model.

A composition comprising viral vectors, non-viral vectors, or acombination of viral and non-viral vectors of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby vectors or viruses are combined in amixture with a pharmaceutically acceptable carrier. As noted above, acomposition, such as phosphate-buffered saline is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient subject. Other suitable carriers are well-knownto those in the art (see, for example, Remington's PharmaceuticalSciences, 9th Ed. (Mack Publishing Co. 1995), and Gilman's thePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985)).

For purposes of therapy, a therapeutic gene expression vector, or arecombinant virus comprising such a vector, and a pharmaceuticallyacceptable carrier are administered to a subject in a therapeuticallyeffective amount. A combination of an expression vector (or virus) and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient subject. In the present context, an agent is physiologicallysignificant if its presence inhibits the growth of tumor cells orinhibits viral infection. An inhibition of tumor growth may beindicated, for example, by a decrease in the number of tumor cells,decreased metastasis, a decrease in the size of a solid tumor, orincreased necrosis of a tumor. Indicators of viral infection inhibitioninclude decreased viral titer, a decrease in detectable viral antigen,or an increase in anti-viral antibody titer.

When the subject treated with a therapeutic gene expression vector or arecombinant virus is a human, then the therapy is preferably somaticcell gene therapy. That is, the preferred treatment of a human with atherapeutic gene expression vector or a recombinant virus does notentail introducing into cells a nucleic acid molecule that can form partof a human germ line and be passed onto successive generations (i.e.,human germ line gene therapy).

14. Production of Transgenic Mice

Transgenic mice can be engineered to over-express the Zcyto13 gene inall tissues or under the control of a tissue-specific ortissue-preferred regulatory element. These over-producers of Zcyto13 canbe used to characterize the phenotype that results from over-expression,and the transgenic animals can serve as models for human disease causedby excess Zcyto 13. Transgenic mice that over-express Zcyto13 alsoprovide model bioreactors for production of Zcyto13 in the milk or bloodof larger animals. Methods for producing transgenic mice are well-knownto those of skill in the art (see, for example, Jacob, “Expression andKnockout of Interferons in Transgenic Mice,” in Overexpression andKnockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124(Academic Press, Ltd. 1994), Monastersky and Robl (eds.), Strategies inTransgenic Animal Science (ASM Press 1995), and Abbud and Nilson,“Recombinant Protein Expression in Transgenic Mice,” in Gene ExpressionSystems: Using Nature for the Art of Expression, Fernandez and Hoeffler(eds.), pages 367-397 (Academic Press, Inc. 1999)).

For example, a method for producing a transgenic mouse that expresses aZcyto13 gene can begin with adult, fertile males (studs) (B6C3fl, 2-8months of age (Taconic Farms, Germantown, N.Y.)), vasectomized males(duds) (B6D2fl, 2-8 months, (Taconic Farms)), prepubescent fertilefemales (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertilefemales (recipients) (B6D2fl, 2-4 months, (Taconic Farms)). The donorsare acclimated for one week and then injected with approximately 8IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company;St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of humanChorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.Donors are mated with studs subsequent to hormone injections. Ovulationgenerally occurs within 13 hours of hCG injection. Copulation isconfimed by the presence of a vaginal plug the morning following mating.

Fertilized eggs are collected under a surgical scope. The oviducts arecollected and eggs are released into urinanalysis slides containinghyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (described, for example, by Menino andO'Claray, Biol. Reprod 77:159 (1986), and Dienhart and Downs, Zygote4:129 (1996)) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂ at37° C. The eggs are then stored in a 37° C./5% CO₂ incubator untmicroinjection.

Ten to twenty micrograms of plasmid DNA containing a Zcyto13 encodingsequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl(pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10nanograms per microliter for microinjection. For example, the Zcyto13encoding sequences can encode amino acid residues 22 to 199 of SEQ IDNO:2.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfuillymicroinjected eggs are transferred into an organ tissue-culture dishwith pre-gassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, two-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy two-cell embryos fromthe previous day's injection are transferred into the recipient. Theswollen ampulla is located and holding the oviduct between the ampullaand the bursa, a nick in the oviduct is made with a 28 g needle close tothe bursa, making sure not to tear the ampulla or the bursa.

The pipette is trasferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductiveorgans allowed to slide in. The peritoncal wall is closed with onesuture and the skin closed with a wound clip. The mice recuperate on a37° C. slide warmer for a minimum of four hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using, for example, a QIAGENDNEASY kit following the manufacturer's instructions. Genomic DNA isanalyzed by PCR using primers designed to amplify a Zcyto13 gene or aselectable marker gene that was introduced in the same plasmid.Illustrative primers suitable for amplifying Zcyto13 are described inthe Examples, below. After animals are confirmed to be transgenic, theyare back-crossed into an inbred strain by placing a transgenic femalewith a wild-type male, or a transgenic male with one or two wild-typefemale(s). As pups are born and weaned, the sexes are separated, andtheir tails snipped for genotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the zyphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum and the left lateral lobe ofthe liver exteriorized. Using 4-0 silk, a tie is made around the lowerlobe securing it outside the body cavity. An atraumatic clamp is used tohold the tie while a second loop of absorbable Dexon (American Cyanamid;Wayne, N.J.) is placed proximal to the first tie. A distal cut is madefrom the Dexon tie and approximately 100 mg of the excised liver tissueis placed in a sterile petri dish. The excised liver section istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage placed on a37° C. heating pad for 24 hours post operatively. The animal is checkeddaily post operatively and the wound clips removed 7-10 days aftersurgery. The expression level of Zcyto13 mRNA is examined for eachtransgenic mouse using an RNA solution hybridization assay or polymerasechain reaction.

This general approach was used to produce transgenic mice comprising aZcyto13 expression vector. The vector contained a Zcyto13 gene that wassynthesized with PCR primers ZC22243 (5′ CGT ACG GGC CGG CCA CCA TGA CTCCAA AGT TT 3′; SEQ ID NO:14) and ZC22244 (5′ CGC GCG GGC GCG CCC TAT TTAAGT TCT TGC TT 3′; SEQ ID NO:15). A preliminary study was performed witha male transgenic mouse, which expressed Zcyto13 in its liver. Flowcytometry analysis of spleen- and bone marrow-derived cells indicatedthat Zcyto13 may affect the development of the myeloid lineage in bonemarrow.

In addition to producing transgenic mice that over-express Zcyto13, itis useful to engineer transgenic mice with either abnormally low or noexpression of the gene. Such transgenic mice provide useful models fordiseases associated with a lack of Zcyto13. As discussed above, Zcyto13gene expression can be inhibited using anti-sense genes, ribozyme genes,or external guide sequence genes. To produce transgenic mice thatunder-express the Zcyto13 gene, such inhibitory sequences are targetedto Zcyto13 mRNA. Methods for producing transgenic mice that haveabnormally low expression of a particular gene are known to those in theart (see, for example, Wu et al., “Gene Underexpression in CulturedCells and Animals by Antisense DNA and RNA Strategies,” in Methods inGene Biotechnology, pages 205-224 (CRC Press 1997)).

An alternative approach to producing transgenic mice that have little orno Zcyto13 gene expression is to generate mice having at least onenormal Zcyto13 allele replaced by a nonfuinctional Zcyto13 gene. Onemethod of designing a nonfunctional Zcyto13 gene is to insert anothergene, such as a selectable marker gene, within a nucleic acid moleculethat encodes Zcyto13. Standard methods for producing these so-called“knockout mice” are known to those skilled in the art (see, for example,Jacob, “Expression and Knockout of Interferons in Transgenic Mice,” inOverexpression and Knockout of Cytokines in Transgenic Mice, Jacob(ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al., “NewStrategies for Gene Knockout,” in Methods in Gene Biotechnology, pages339-365 (CRC Press 1997)).

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which is provided by wayof illustration and is not intended to be limiting of the presentinvention.

EXAMPLE 1 Construction of a Nucleic Acid Molecule Encoding Zcyto13

5′ RACE was performed to obtain a 5′ sequence from an expressed sequencetag. The 5′ RACE was performed as follows: 3 μl of 1/100 dilutedplacenta marathon cDNA, 20 pmoles each of oligonucleotide primers ZC9739(5′ CCA TCC TAA TAC GAC TCA CTA TAG GGC 3′; SEQ ID NO:5) and ZC20980 (5′ACT GCA CTG CCT TGA ACC CTG A 3′; SEQ ID NO:6), and 1 U of mixture ofExTaq/Taq antibody (1:1) were combined in 25 μl reactions. The reactionswere run as follows: 94° C. for 2 minutes, then 30 cycles of 94° C. for20 seconds, 64° C. for 30 seconds, 72° C. for 45 seconds, and thereaction was stopped with a 2 minute incubation at 72° C. One microlitereach of 1/30 diluted first PCR product was used as template for a nestedPCR. Twenty picomoles each of oligonucleotide primers ZC9719 (5′ ACT CACTAT AGG GCT CGA GCG GC 3′; SEQ ID NO:7) and ZC20981 (5′ GTT CTT GCT TGAAGG TGG GTG ATT 3′; SEQ ID NO:8) and 1 U of mixture of ExTaq/Taqantibody (1:1) were combined in 25 μl reactions. The reactions were runas follows: 94° C. for 2 minutes, then 35 cycles of 94° C. for 20seconds, 63° C. for 30 seconds, 72° C. for 45 seconds, and the reactionwas stopped with a 4 minute incubation at 72° C. The PCR products werefractionated on an agarose gel. Obvious bands were obtained in placenta,purified with QlAquick (QIAGEN Inc.; Valencia, Calif.) and ligated intoa pCR2.1 vector using TA cloning kit (Invitrogen). Clones with insertswere sequenced, and the results indicated that the 5′ RACE productcontained the 5′ stop codon, first Met and signal peptide informationfor a fill length cDNA.

New primers ZC21590 (5′ ATA CTA AGC ACC AGG GTT GAG AAT G 3′; SEQ IDNO:9) and ZC21591 (5′ AGG TAG CAT TAG CAG CAT CCT GGT A 3′; SEQ IDNO:10) were designed according to the new 5′ sequence information on theclone. PCR was used to amplify entire coding sequence with the primerpair above, and RNA samples from placenta, testis and 7d embryo tissueswere used as templates. Three microliters of 1/100 diluted marathoncDNAs, 20 pmoles of each oligonucleotide primers, and 1 U of mixture ofExTaq/Taq antibody (1:1) were used in 25 μl reactions. The reactionswere run as follows: 94° C. for 2 minutes, then 35 cycles of 94° C. for20 seconds, 61° C. for 30 seconds, 72° C. for 30 seconds, and thereactions were stopped with a 4 minute incubation at 72° C. PCR productswere separated on an agarose gel and purified with QIAquick (QIAGENInc.). Purified PCR products were then ligated into pCR2.1 vector usingTA cloning kit (Invitrogen) and inserts were sequenced.

EXAMPLE 2 Expression ofthe Zcyto13 Gene

Northern analyses were performed using a Mouse Multiple Tissue NorthernBlot, a Mouse Embryo Multiple Tissue Northern Blot, and a Mouse RNAMaster Blot (dot blot) (CLONTECH Laboratories, Inc., Palo Alto, Calif.).The hybridization probe was generated from a gel purified PCRamplification product. The probe was made using ZC20979 (5′ CTG ACA GTCTAC CTG GAG TTG GG 3′; SEQ ID NO:11) and ZC20980 as primers and mouseseven-day embryo Marathon Ready cDNA as template. The probe length was310 base pairs. The probe was a radioactively labeled using theREDIPRIME II labeling kit (AMERSHAM PHARMACIA BIOTECH, Inc.; Piscataway,N.J.) according to the manufacturer's protocol. The probe was purifiedusing a NUCTRAP push column (STRATAGENE, La Jolla, Calif.). EXPRESSHYB(CLONTECH) solution was used for the prehybridization and hybridizationsolutions for the Northern blots. Hybridization took place overnight at65° C. Following hybridization, the blots were washed in 2×SSC, 0.1% SDSat room temperature, followed by a wash in 0.1×SSC and 0.1% SDS at 50°C. The blots were exposed to Kodak BioMax film. Bands at 6.5 kilobasesand 2.8 kilobases were clearly visible in heart, brain, liver, kidney,and seven-day embryo on the Northern blots. Faint bands at the same sizewere also visible in lung and testes. The RNA Master Blot showedpositive signals from heart, submaxillary gland, epididymus, andseven-day embryo.

EXAMPLE 3 Southern Analysis of theZcyto13 Gene

Southern analysis was performed using a commercially preparedInterspecies Zoo-Blot from CLONTECH Laboratories, Inc. The Southern blotcontained EcoRI-digested DNA. The hybridization probe was generated asdescribed for Example 2. The probe was a radioactively labeled using theREDIPRIME II labeling kit (AMERSHAM PHARMACIA BIOTECH, Inc.; Piscataway,N.J.) according to the manufacturer's protocol, and the probe waspurified using a NUCTRAP push column (STRATAGENE, La Jolla, Calif.).EXPRESSHYB (CLONTECH) solution was used for the prehybridization andhybridization solutions for the Southern blots. The blot was hybridizedovernight at 65° C., and then, the blots were washed in 2×SSC, 0.1% SDSat room temperature, followed by a wash in 0.1×SSC and 0.1% SDS at 50°C. After washing, the blots were exposed to Kodak BioMax film. The mousegenomic DNA sample contained hybridizing fragments at approximately 5.65and 4.8 kilobases, while the rat sample contained hybridizing fragmentsat approximately 5.2 and 2.1 kilobases.

EXAMPLE 4 Expression of the Zcyto13 Gene Using Adenovirus Constructs

1. Generation of Untagged Zycto13 Recombinant Adenovirus

The protein coding region of murine Zcyto13 was amplified by PCR usingprimers that added FseI and AscI restriction sties at the 5′ and 3′termini respectively. PCR primers ZC21924 (5′ CAC ACA GGC CGG CCA CCATGA CTC CAA AGT TTT TAT GGC 3′; SEQ ID NO:12) and ZC21923 (5′ CAC ACAGGC GCG CCT CTA TTT AAG TTC TTG CTT GAA GGT GGG 3′; SEQ ID NO:13) wereused with template pCR2.1 plasmid containing the full-length murineZcyto13 cDNA in a PCR reaction as follows: one cycle at 95° C. for 5minutes, followed by 15 cycles at 95° C. for 0.5 minute, 58° C. for 0.5minute, and 72° C. for 0.5 minute, followed by 72° C. for 7 minutes,followed by a 4° C. soak. The PCR reaction product was loaded onto a1.2% (low melt) SEAPLAQUE GTG (FMC BioProducts; Rockland, Me.) gel inTAE buffer. The Zcyto13 PCR product was excised from the gel, melted at65°, phenol extracted twice and then ethanol precipitated. The PCRproduct was then digested with FseI-AscI, phenol/chloroform extracted,ethanol precipitated, and rehydrated in 20 μl TE (Tris/EDTA, pH 8).

The 600 base pair Zcyto13 fragment was then ligated into the FseI-AscIsites of a modified pAdTrack CMV (He et al., Proc. Nat'l Acad. Sci. USA95:2509 (1998)). This construct also contains the green fluorescentprotein (GFP) marker gene. The CMV promoter driving GFP expression wasreplaced with the SV40 promoter and the SV40 polyadenylation signal wasreplaced with the human growth hormone polyadenylation signal. Inaddition, the native polylinker was replaced with FseI, EcoRV, and AscIsites. This modified form of pAdTrack CMV was named pZyTrack. Ligationwas performed using the FAST-LINK DNA ligation and screening kit(EPICENTRE TECHNOLOGIES; Madison, Wis.). Clones containing the Zcyto13cDNA were identified by standard mini prep procedures. In order tolinearize the plasmid, approximately 5 μg of the pZyTrack Zcyto13plasmid were digested with PmeI. Approximately 1 μg of the linearizedplasmid was cotransformed with 200 ng of supercoiled pAdEasy (He et al.,Proc. Nat'l Acad. Sci. USA 95:2509 (1998)) into BJ5183 cells. Theco-transformation was performed with a BIO-RAD GENE PULSER (BIO-RADlaboratories, Inc.; Hercules, Calif.) at 2.5 kV, 200 ohms and 25 mFa.The entire co-transformation was plated on four LB plates containing 25μg/ml kanamycin. The smallest colonies were picked and expanded inLB/kanamycin and recombinant adenovirus DNA identified by standard DNAminiprep procedures. Digestion of the recombinant adenovirus DNA withFseI-AscI confirmed the presence of Zcyto13. The recombinant adenovirusminiptep DNA was transformed into DH10B competent cells and DNA preparedusing a QIAGEN maxi prep kit as per kit instructions.

2. Transfection of 293A Cells with Recombinant DNA

Approximately 5 μg of recombinant adenoviral DNA were digested with PacIenzyme for three hours at 37° C. in a reaction volume of 100 μlcontaining 20-30 U of PacI. The digested DNA was extracted twice with anequal volume of phenol/chloroform and precipitated with ethanol. The DNApellet was resuspended in 5 μl distilled water. QBI-293A cells (QuantumBiotechnologies, Inc.; Montreal, Quebec, Canada), inoculated the daybefore and grown to 60-70% confluence in a T25 flask, were transfectedwith the PacI digested DNA. The PacI-digested DNA was diluted up to atotal volume of 50 μl with sterile HBS (150 mM NaCl, 20 mM HEPES). In aseparate tube, 25 μl DOTAP (1 mg/ml; Roche Molecular Biochemicals;Indianapolis, Ind.) were diluted to a total volume of 100 μl with HBS.The DNA was added to the DOTAP, mixed gently by pipeting up and down,and left at room temperature for 15 minutes. The medium was removed fromthe 293A cells and washed with 5 ml serum-free MEMalpha (LIFETECHNOLOGIES, Inc; Rockville, Md.) containing 1 mM sodium pyruvate (LIFETECHNOLOGIES, Inc), 0.1 mM MEM non-essential amino acids (LIFETECHNOLOGIES, Inc) and 25 mM HEPES buffer (LIFE TECHNOLOGIES, Inc). Fivemilliliters of serum-free MEM were added to the 293A cells and held at37° C. The DNA/lipid mixture was added drop-wise to the T25 flask of293A cells, mixed gently and incubated at 37° C. for 4 hours. After fourhours, the medium containing the DNA/lipid mixture was aspirated, andreplaced with 5 ml complete MEM containing 5% fetal bovine serum. Thetransfected cells were monitored for green fluorescent protein (GFP)expression and formation of foci.

Seven days after transfection of 293A cells with the recombinantadenoviral DNA, the cells expressed the GFP protein and started to formfoci. These foci are viral “plaques.” The crude viral lysate wascollected using a cell scraper to collect all of the 293A cells. Thelysate was transferred to a 50 ml conical tube. To release most of thevirus particles from the cells, three freeze/thaw cycles were performedin a dry ice/ethanol bath and a 37° water bath.

3. Amplification of Recombinant Adenovirus (rAdV)

The crude lysate was amplified (“primary amplification”) to obtain aworking stock of Zcyto13 rAdV lysate. Two hundred milliliters of cruderAdV lysate were added to each of ten 10 cm plates of nearly confluent(80-90%) 293A cells, which had been set up 20 hours previously. Theplates were monitored for 48 to 72 hours for cytopathic effect under thewhite light microscope and expression of GFP under the fluorescentmicroscope. When all of the 293A cells showed cytopathic effect, thisprimary amplification stock lysate was collected and freeze/thaw cyclesperformed as described above.

Secondary amplification of Zcyto13 rAdV was obtained as follows. Twenty15 cm tissue culture dishes of 293A cells were prepared so that thecells were 80-90% confluent. All but 20 milliliters of 5% MEM media wasremoved, and each dish was inoculated with 300-500 ml primary amplifiedrAdv lysate. After 48 hours, the 293A cells were lysed from virusproduction and this lysate was collected into 250 ml polypropylenecentrifuge bottles and the rAdV purified.

4. AdV/cDNA Purification

NP40 detergent was added to a final concentration of 0.5% to the bottlesof crude lysate to lyse all cells. Bottles were placed on a rotatingplatform for 10 minutes, agitating as fast as possible withoutdisplacing the bottles. The debris was pelleted by centrifugation at20,000×g for 15 minutes. The supernatant was transferred to 250 mlpolycarbonate centrifuge bottles, and 0.5 volume of 20% PEG8000/2.5 MNaCl solution was added. The bottles were shaken overnight on ice. Thebottles were centrifuged at 20,000×g for 15 minutes and supernatantdiscarded into a bleach solution. The precipitated virus/PEG appeared asa white precipitate located in two vertical lines along the wall of thebottle on either side of the spin mark. Using a sterile cell scraper,the precipitate from two bottles was resuspended in 2.5 ml PBS. Thevirus solution was placed in 2 ml microcentrifuge tubes and centrifugedat 14,000×g in the microfuge for 10 minutes to remove any additionalcell debris. The supernatant from the 2 ml microcentrifuge tubes wastransferred into a 15 ml polypropylene snapcap tube and adjusted to adensity of 1.34 g/ml with cesium chloride (CsCl). The volume of thevirus solution was estimated and 0.55 g/ml of was CsCl added. The CsClwas dissolved and 1 ml of this solution weighed 1.34 g. The solution wastransferred to polycarbonate thick-walled centrifuge tubes, andcentrifuged at 80,000 rpm (348,000×g) for 3-4 hours at 25° C. in aBeckman Optima TLX micro-ultracentrifuge with the TLA-100.4 rotor. Thevirus formed a white band. Using wide-bore pipette tips, the virus bandwas collected.

The virus from the gradient has a large amount of CsCl which must beremoved before it can be used with cells. Pharmacia PD-10 columnsprepacked with SEPHADEX G-25M (Amersham Pharnacia Biotech, Inc;Piscataway, N.J.) were used to desalt the virus preparation. The columnwas equilibrated with 20 ml of PBS. The virus was loaded and allowed torun into the column. Five milliliters of PBS were added to the columnand fractions of 8-10 drops collected. The optical densities of 1:50dilutions of each fraction were determined at 260 nm on aspectrophotometer. A clear absorbance peak was present between fractions7-12. These fractions were pooled and the optical density (OD) of a 1:25dilution determined. The following formula was used to convert OD intovirus concentration: (OD at 260 nm)(25)(1.1×10¹²)=virions/ml. The OD ofa 1:25 dilution of the Zcyto13 rAdV was 0.059, giving a virusconcentration of 4.9×10¹² virions/ml.

To store the virus, glycerol was added to the purified virus to a finalconcentration of 15%, mixed gently but effectively, and stored inaliquots at −80° C.

5. Viral Titration Assay

A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Quebec,Canada) was followed to measure recombinant virus infectivity. Briefly,two 96-well tissue culture plates were seeded with 1×10⁴293A cells perwell in MEM containing 2% fetal bovine serum for each recombinant virusassayed. After 24 hours, 10-fold dilutions of each virus from 1×10⁻² to1×10⁻¹⁴ were made in MEM containing 2% fetal bovine serum. One hundredmicroliters of each dilution were placed in each of 20 wells. After fivedays at 37° C., wells were read either positive or negative forcytopathic effect, and a value for “plaque forming units/ml” (PFU) iscalculated.

The tissue culture infectious dose at 50% cytopathic effect (TCID₅₀) wasproduced as per Quantum Biotecbnologies, Inc., above. The titer isdetermined from a plate in which virus is diluted from 10⁻² to 10⁻¹⁴,and read five days after the infection. At each dilution a ratio (R) ofpositive wells for cytopathic effect per the total number of wells isdetermined.

To calculate the titer of the undiluted virus sample, factor “F” wasfirst calculated, as I+d(S-0.5), where “S” is the sum of the ratios (R),and “d” is log₁₀ of the dilution series (e.g., “d” is equal to one for aten-fold dilution series). The titer of the undiluted sample iscalculated as: 10^((1+F))=TCID₅₀/ml. To convert TCID₅₀/ml to pfu/ml, 0.7is subtracted from the exponent in the calculation for titer (T). Usingthis method, the Zcyto13 adenovirus had a titer of 4.0×10⁹ pfu/ml.

EXAMPLE 5 Biological Activity of Zcyto13 Protein

1. Stimulation of Expression from an Interferon-Responsive Promoter

In one series of experiments, conditioned medium (CM) containingZcyto13m protein was generated by infecting 293A cells with recombinantadenovirus containing the cDNA for Zcyto13m (AdZy-zcyto13m) at amultiplicity of infection of 400 particles per cell. CM was harvested attime points between 40 hours post infection and stored at −20° C. CM wasalso generated from an infection with a recombinant adenovirus lacking acDNA (AdZy-parental). Prior to use, a portion of the CM was concentrated14 fold in a Millipore Ultrafree-15 (5,000 nominal molecular weightlimit) centrifugal filter, and then, filtered through a MilliporeUltrafree-15 (100,000 nominal molecular weight limit) centrifugal filterto reduce the amount of viral particles present in the media, andfinally filtered through a Millipore 0.2 μm, syringe filter to sterilizethe CM. Concentrated CM samples were diluted 1:2 in binding, buffer andincubated with cells from a murine cell line for 5 hours at 37° C. CMcontaining Zcyto13 protein stimulated a 17-fold increase in geneexpression from an interferon-responsive promoter, while control CM,appeared to stimulate gene expression by 1.4 fold. These resultsindicate that Zcyto13 induces expression from an interferon-responsivepromoter.

2. Anti-Viral Activity of Zcyto13

Another series of experiments examined the anti-viral activity ofZcyto13. In these studies, the anti-viral assay was performed by platingL929 cells (ATCC No. CCL-1) in growth media RPMI medium 1640 containing10% fetal bovine serum, penicillin, streptomycin, and L-glutamine in96-well format at 50,000 cells per well. Adenovirus CM from 293A cellsinfected with either AdZy-zcyto13m or AdZy-parental, as described above,was incubated with cells overnight. A positive control in the assay wasprovided by murine interferon-α serially diluted 1:10, starting at 100ng/ml. L929 cells with growth media alone provided the negative control.Treated cells were incubated for 24 hours. The media were discarded,fresh medium was added, and encephalomyocarditis virus (ATCC No. vr129b)was introduced at a multiplicity of infection of 0.1 (i.e., one virusparticle for every ten L929 cells). The cells were incubated in thepresence of the virus for 24 hours, and then, the wells were scored forpercent cytopathic effect (CPE). As shown in Table 5, conditioned mediumcontaining Zcyto13 effectively inhibited viral infection of the cells.

TABLE 5 Treatment Observed Cytopathic Effect medium alone noneencephalomyocarditis virus (EMCV) >90% interferon-α + EMCV noneAdZy-parental + EMCV >80% AdZy-Zcyto13 + EMCV none

3. Mediation of Biological Activity Through the Human InterferonReceptor

Baf3, an interleukin-3 dependent pre-B cell line, was transfected withtwo chimeric receptors containing the extracellular domains of humaninterferon receptor and the signaling domains of murine MPL. Thepresence of the interferon-α/murine MPL and human interferon-β/murineMPL receptors was selected with zeocin (at 2 mg/ml) and puromycin (at 2μg/ml).

Baby hamster kidney (BHK) cells were stably transfected with anexpression vector containing the CMV promoter plus intron A upstream ofeither the murine Zcyto13 cDNA, or an unrelated cDNA, encoding “Zα30,”using BRL lipofectamine. Stably transfected cells were seeded in a cellfactory with serum free medium and allowed to grow for three days beforeconditioned media were harvested and concentrated in a 5K filter to 10×.Concentrated conditioned medium was stored at 4° C.

The assay to test for proliferation of Baf3 cells via signaling throughthe chimeric receptor was performed as follows. In a 96 well plate eight1:2 serial dilutions of growth medium alone (RPMI 1640, 10% fetal bovineserum, 1 mM sodium pyruvate, 2 mM L-glutamine), murine interleukin-3(starting at 50 pg/ml in growth medium), human interferon-α (starting at100 ng/ml in growth medium), human interferon-β (starting at 100 ng/mlin growth medium), murine interferon-α (starting at 100 ng/ml in growthmedium), murine interferon-β (starting at 100 ng/ml in growth medium),murine Zcyto13 (starting at 5×in assay) with and without anti-humaninterferon receptor chain 2 at 5 μg/ml per well (diluted in growthmedium), and murine Zα30 (starting at 5×in assay). The final volume ofeach dilutions was 100 μl.

The Baf3 parental cell line and Baf3 cells transfected with humaninterferon/murine MPL receptor were washed three times in growth media(see above), pellets were resuspended in growth medium, and cells werecounted and diluted in growth media to 5,000 cells/100 μl. One hundredmicroliters of diluted cells were added to each dilution of samples. Theassay plate was incubated in a 37° C. incubator for three to four days.A 20 μl aliquot of Alomar blue was added to each well and the plate wasincubated overnight at 37° C. The plates were read on the fluorescentplate reader at excitation wavelength of 544 and emission wavelength590. The data demonstrated that interferon-α and -β are able tostimulate the Baf3 cells transfected with the chimeric receptors toproliferate. Zcyto13 was also able to stimulate proliferation in thetransfected Baf3 cells, thus demonstrating that Zcyto13 signals throughthe human interferon receptor.

4. Antiproliferation Assay Using a BAF3 Cell Line

Baf3 was used to determine if Zcyto13 has anti-proliferative properties.Baby hamster kidney (BHK) cells were stably transfected with anexpression vector containing the CMV promoter plus intron A upstream ofthe murine Zcyto13 cDNA or an unrelated cDNA, called Zα30, using BRLlipofectamine. Stably transfected cells were seeded in a cell factory inserum free media and allowed to grow for three days before conditionedmedia was harvested and concentrated in a 5K filter to 10×. Concentratedconditioned medium samples were stored at 4° C.

The following assay was used to test for anti-proliferation of Baf3. Ina 96 well plate, eight 1:2 serial dilutions were made of growth mediaalone (RPMI 1640, 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mML-glutamine), or murine IL-3 (starting at 50 μg/ml in growth medium)with final volume of 100 μl. Fifty microliters of the following wereadded to both growth media alone or mIL-3 diluted lanes: humaninterferon-α (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium),human interferon-β (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in growthmedium), murine interferon-α (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted ingrowth medium), murine interferon-β (100 ng/ml, 10 ng/ml, or 1 ng/mldiluted in growth medium), murine Zcyto13 (at 2.5×, 0.5×, or 0.1×), andmurine Zα30 (at 2.5×, 0.5×, or 0.1×).

The Baf3cell ine was washed three times in growth medium, pellets wereresuspended in growth medium, cells were counted and diluted in growthmedium to 5,000 cells/50 μl. Fifty microliters of diluted cells werethen added to each dilution of samples. Assay plates were incubated in a37° C. incubator for three to four days. Twenty microliters of Alomarblue were then added to each well and the plate were incubated overnightat 37° C. The plates were read on the fluorescent plate reader atexcitation wavelength of 544 and emission wavelength 590. The resultsdemonstrated that α-interferon and Zcyto13 inhibited proliferation ofBaf3 cells.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

22 1 768 DNA mouse CDS (23)...(619) 1 atactaagca ccagggttga ga atg actcca aag ttt tta tgg ctg gtg gcc 52 Met Thr Pro Lys Phe Leu Trp Leu ValAla 1 5 10 ctt gtg gct cta tac att ccg ccc atc caa tct ctg aac tgt gtttac 100 Leu Val Ala Leu Tyr Ile Pro Pro Ile Gln Ser Leu Asn Cys Val Tyr15 20 25 ctg gat gat agc atc ttg gaa aat gtg aaa ctt ctg ggc agt acc atg148 Leu Asp Asp Ser Ile Leu Glu Asn Val Lys Leu Leu Gly Ser Thr Met 3035 40 acc ggc ttt ccc tta aga tgt cta aaa gat atc aca gat ttt aag ttt196 Thr Gly Phe Pro Leu Arg Cys Leu Lys Asp Ile Thr Asp Phe Lys Phe 4550 55 cct aaa gag att ttg cca tac atc cag cat atg aaa agg gag ata aac244 Pro Lys Glu Ile Leu Pro Tyr Ile Gln His Met Lys Arg Glu Ile Asn 6065 70 gcc gtc tcc tat cgt ata tcc tct ctg gca cta act atc ttc aat ctt292 Ala Val Ser Tyr Arg Ile Ser Ser Leu Ala Leu Thr Ile Phe Asn Leu 7580 85 90 aaa ggc tcc atc cct cca gtg aca gag gaa cac tgg gaa cgt atc aga340 Lys Gly Ser Ile Pro Pro Val Thr Glu Glu His Trp Glu Arg Ile Arg 95100 105 tcg gga ctt ttc aaa caa gtg cgg caa gct caa gag tgc ttc atg gac388 Ser Gly Leu Phe Lys Gln Val Arg Gln Ala Gln Glu Cys Phe Met Asp 110115 120 gag gag aaa gag aac agg gaa cat cct cac tcc gag gac ttc ctg aca436 Glu Glu Lys Glu Asn Arg Glu His Pro His Ser Glu Asp Phe Leu Thr 125130 135 gtc tac ctg gag ttg ggc aag tat ttc ttc aga atc aaa aag ttc ctg484 Val Tyr Leu Glu Leu Gly Lys Tyr Phe Phe Arg Ile Lys Lys Phe Leu 140145 150 ata aat aag aaa tac agt ttc tgt gca tgg aag att gtc aca gtg gaa532 Ile Asn Lys Lys Tyr Ser Phe Cys Ala Trp Lys Ile Val Thr Val Glu 155160 165 170 ata aga aga tgt ttc att ata ttt tcc aag tcc aga aaa cta ctcaaa 580 Ile Arg Arg Cys Phe Ile Ile Phe Ser Lys Ser Arg Lys Leu Leu Lys175 180 185 atg ata tca gaa tca ccc acc ttc aag caa gaa ctt aaatagaagctgc 629 Met Ile Ser Glu Ser Pro Thr Phe Lys Gln Glu Leu Lys 190195 aattgctcaa atgtctccaa gaacgcttta ttctaaagcc attaccagga tgctgctaat689 gctaccttca gatgcaagac ttttcaagtt cagggttcaa ggcagtgcag tcaaagaaag749 tcttaagcaa aagatgaac 768 2 199 PRT mouse 2 Met Thr Pro Lys Phe LeuTrp Leu Val Ala Leu Val Ala Leu Tyr Ile 1 5 10 15 Pro Pro Ile Gln SerLeu Asn Cys Val Tyr Leu Asp Asp Ser Ile Leu 20 25 30 Glu Asn Val Lys LeuLeu Gly Ser Thr Met Thr Gly Phe Pro Leu Arg 35 40 45 Cys Leu Lys Asp IleThr Asp Phe Lys Phe Pro Lys Glu Ile Leu Pro 50 55 60 Tyr Ile Gln His MetLys Arg Glu Ile Asn Ala Val Ser Tyr Arg Ile 65 70 75 80 Ser Ser Leu AlaLeu Thr Ile Phe Asn Leu Lys Gly Ser Ile Pro Pro 85 90 95 Val Thr Glu GluHis Trp Glu Arg Ile Arg Ser Gly Leu Phe Lys Gln 100 105 110 Val Arg GlnAla Gln Glu Cys Phe Met Asp Glu Glu Lys Glu Asn Arg 115 120 125 Glu HisPro His Ser Glu Asp Phe Leu Thr Val Tyr Leu Glu Leu Gly 130 135 140 LysTyr Phe Phe Arg Ile Lys Lys Phe Leu Ile Asn Lys Lys Tyr Ser 145 150 155160 Phe Cys Ala Trp Lys Ile Val Thr Val Glu Ile Arg Arg Cys Phe Ile 165170 175 Ile Phe Ser Lys Ser Arg Lys Leu Leu Lys Met Ile Ser Glu Ser Pro180 185 190 Thr Phe Lys Gln Glu Leu Lys 195 3 597 DNA ArtificialSequence This degenerate sequence encodes the amino acid sequence of SEQID NO2. 3 atgacnccna arttyytntg gytngtngcn ytngtngcny tntayathccnccnathcar 60 wsnytnaayt gygtntayyt ngaygaywsn athytngara aygtnaarytnytnggnwsn 120 acnatgacng gnttyccnyt nmgntgyytn aargayatha cngayttyaarttyccnaar 180 garathytnc cntayathca rcayatgaar mgngaratha aygcngtnwsntaymgnath 240 wsnwsnytng cnytnacnat httyaayytn aarggnwsna thccnccngtnacngargar 300 caytgggarm gnathmgnws nggnytntty aarcargtnm gncargcncargartgytty 360 atggaygarg araargaraa ymgngarcay ccncaywsng argayttyytnacngtntay 420 ytngarytng gnaartaytt yttymgnath aaraarttyy tnathaayaaraartaywsn 480 ttytgygcnt ggaarathgt nacngtngar athmgnmgnt gyttyathathttywsnaar 540 wsnmgnaary tnytnaarat gathwsngar wsnccnacnt tyaarcargarytnaar 597 4 16 PRT Artificial Sequence Peptide linker 4 Gly Gly SerGly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 5 27 DNAArtificial Sequence PCR primer 5 ccatcctaat acgactcact atagggc 27 6 22DNA Artificial Sequence PCR primer 6 actgcactgc cttgaaccct ga 22 7 23DNA Artificial Sequence PCR primer 7 actcactata gggctcgagc ggc 23 8 24DNA Artificial Sequence PCR primer 8 gttcttgctt gaaggtgggt gatt 24 9 25DNA Artificial Sequence PCR primer 9 atactaagca ccagggttga gaatg 25 1025 DNA Artificial Sequence PCR primer 10 aggtagcatt agcagcatcc tggta 2511 23 DNA Artificial Sequence PCR primer 11 ctgacagtct acctggagtt ggg 2312 39 DNA Artificial Sequence PCR primer. 12 cacacaggcc ggccaccatgactccaaagt ttttatggc 39 13 42 DNA Artificial Sequence PCR primer. 13cacacaggcg cgcctctatt taagttcttg cttgaaggtg gg 42 14 32 DNA ArtificialSequence PCR primer. 14 cgtacgggcc ggccaccatg actccaaagt tt 32 15 32 DNAArtificial Sequence PCR primer. 15 cgcgcgggcg cgccctattt aagttcttgc tt32 16 15 PRT Artificial Sequence Amino acid motif. 16 Xaa Xaa Xaa XaaXaa Leu Xaa Xaa Xaa Xaa Asp Phe Xaa Xaa Pro 1 5 10 15 17 19 PRTArtificial Sequence Amino acid motif. 17 Xaa Xaa Leu Xaa Xaa Xaa Lys XaaXaa Xaa Xaa Ala Trp Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Glu 18 9 DNAArtificial Sequence Nucleotide sequence. 18 atgcacggg 9 19 9 DNAArtificial Sequence Nucleotide sequence. 19 cccgtgcat 9 20 9 DNAArtificial Sequence Nucleotide sequence. 20 atggagctt 9 21 9 DNAArtificial Sequence Nucleotide sequence. 21 agcttgagt 9 22 9 DNAArtificial Sequence Nucleotide sequence. 22 tcgactacc 9

We claim:
 1. An isolated nucleic acid molecule, wherein the nucleic acidmolecule encodes the amino acid sequence of SEQ ID NO:2.
 2. The isolatednucleic acid molecule of claim 1, wherein the nucleic acid moleculecomprises the nucleotide sequence of nucleotides 23 to 619 of SEQ IDNO:1.
 3. An isolated nucleic acid molecule that encodes a polypeptidehaving an amino acid sequence that is a variant of the amino acidsequence of SEQ ID NO:2, wherein any difference between the amino acidsequence encoded by the nucleic acid molecule and the correspondingamino acid sequence of SEQ ID NO:2 is due to a conservative amino acidsubstitution and wherein the encoded polypeptide exhibits anti-viral oranti-proliferative activity.
 4. The isolated nucleic acid molecule ofclaim 3, wherein the amino acid sequence encoded by the nucleic acidmolecule is characterized by at least one amino acid substitution withinSEQ ID NO:2 selected from the group consisting of: (a) an alanine forglycine³⁹, (b) a valine for leucine⁶³, (c) a threonine for serine⁸¹, (d)a valine for isoleucine¹⁰⁵, and (e) a leucine for valine¹¹³.
 5. Theisolated nucleic acid molecule of claim 3, wherein the nucleic acidmolecule encodes a polypeptide that exhibits anti-viral activity.
 6. Anisolated nucleic acid molecule that encodes a polypeptide comprisingamno acid residues 22 to 188 of SEQ ID NO:2.
 7. The isolated nucleicacid molecule of claim 6, comprising the nucleotide sequence ofnucleotides 86 to 586 of SEQ ID NO:1.
 8. The isolated nucleic acidmolecule of claim 6, wherein the polypeptide comprises amino acidresidues 22 to 199 of SEQ ID NO:2.
 9. The isolated nucleic acid moleculeof claim 8, comprising the nucleotide sequence of nucleotides 86 to 619of SEQ ID NO:1.
 10. A vector, comprising the isolated nucleic acidmolecule of claim
 3. 11. A vector, comprising the isolated nucleic acidmolecule of claim
 6. 12. A vector, comprising the isolated nucleic acidmolecule of claim
 8. 13. An expression vector, comprising the nucleicacid molecule of claim 8, a transcription promoter, and a transcriptionterminator, wherein the promoter is operably linked with the nucleicacid molecule, and wherein the nucleic acid molecule is operably linkedwith the transcription terminator.
 14. A recombinant host cellcomprising the expression vector of claim 13, wherein the host cell isselected from the group consisting of bacterium, yeast cell, fungalcell, insect cell, avian cell, mammalian cell, and plant cell.
 15. Therecombinant host cell of claim 14, wherein the host cell is a bacterium.16. The recombinant host cell of claim 14, wherein the host cell is ayeast cell.
 17. The recombinant host cell of claim 14, wherein the hostcell is a fungal cell.
 18. The recombinant host cell of claim 14,wherein the host cell is a insect cell.
 19. The recombinant host cell ofclaim 14, wherein the host cell is a avian cell.
 20. The recombinanthost cell of claim 14, wherein the host cell is a mammalian cell. 21.The recombinant host cell of claim 14, wherein the host cell is a plantcell.
 22. A method of producing a polypeptide that comprises amino acidresidues 22 to 199 of SEQ ID NO:2, the method comprising the step ofculturing recombinant host cells that comprise the expression vector ofclaim 13, and that produce the polypeptide.
 23. The method of claim 22,further comprising the step of isolating the polypeptide from thecultured recombinant host cells.